Compounds and Method of Identifying, Synthesizing, Optimizing and Profiling Protein Modulators

ABSTRACT

This invention relates to methods of identifying, synthesizing, optimizing and profiling compounds that are inhibitors or activators of proteins, both naturally occurring endogenous proteins as well as certain variant forms of endogenous proteins, and novel methods of identifying such variants. The method accelerates the identification and development of compounds as potential therapeutically effective drugs by simplifying the pharmaceutical discovery and creation process through improvements in hit identification, lead optimization, biological profiling, and rapid elimination of toxic compounds. Implementation results in overall cost reductions in the drug discovery process resulting from the corresponding increases in efficiency.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 13/873,740 filed Apr.30, 2013, which is a continuation of U.S. Ser. No. 11/604,109, now U.S.Pat. No. 8,431,110, which is a continuation-in-part of PCT InternationalApplication PCT/US06/33890, filed Aug. 29, 2006, which is a continuationin part of PCT/US2005/18412, filed May 23, 2005, and claims priority toU.S. Ser. No. 60/739,477, filed Nov. 23, 2005, U.S. Ser. No. 60/739,476,filed Nov. 23, 2005, U.S. Ser. No. 60/741,767, filed Dec. 2, 2005, U.S.Ser. No. 60/751,030, filed Dec. 16, 2005, U.S. Ser. No. 60/783,106,filed Mar. 13, 2006, U.S. Ser. No. 60/785,904, filed Mar. 23, 2006, U.S.Ser. No. 60/785,817, filed Mar. 23, 2006, and U.S. Ser. No. 60/789,379,filed Apr. 4, 2006.

FIELD OF THE INVENTION

This invention relates to methods of identifying, synthesizing,optimizing and profiling compounds that are inhibitors or activators ofproteins, both naturally occurring endogenous proteins as well ascertain variant forms of endogenous proteins, and novel methods ofidentifying such variants. The method accelerates the identification anddevelopment of compounds as potential therapeutically effective drugs bysimplifying the pharmaceutical discovery and creation process throughimprovements in hit identification, lead optimization, biologicalprofiling, and rapid elimination of toxic compounds. Implementationresults in overall cost reductions in the drug discovery processresulting from the corresponding increases in efficiency.

BACKGROUND OF THE INVENTION

Important components of modern new drug discovery/creation methods thatare directed towards a selected protein target present in a human cellinclude:

1. identification of“hit” compounds which inhibit or activate theselected target protein. (A hit is defined for these purposes as acompound that scores positively in a given assay and may posess some ofthe effects and pharmacological properties that the investigatordesires. In modern pharmaceutical research, however, hits are virtuallynever final clinical candidates without substantial furthermodification);

2. selection of a lead compound upon which to base further studies andrefinements of the initial hit compound;

3. optimization of a lead compound (whose chemical structure is eitherrelated to or identical to the original hit compound) by making a seriesof chemical modifications designed primarily to improve the inhibitoryor activating properties of the lead compound with respect to the targetprotein, but which may also improve bioavailability, plasma half-life,or reduce toxicity;

4. profiling the spectrum of biological activity of a given leadcompound (including an optimized lead) in order to determine itsrelative specificity and selectivity for the chosen target protein ascompared to other non-target proteins, some of which may be closelyrelated to the target protein itself (such as other members of a proteinfamily);

5. preclinical in-vitro and in-vivo animal studies designed to evaluatedosing ranges, carcinogenicity, absorption, distribution, metabolism,excretion, pharmakokinetics, oral bioavailability (if desired),pharmacodynamics, toxicity, and related parameters;

6. clinical trials in healthy volunteers and in patients afflicted withthe disease for which the potential therapeutic treatment is thought tobe beneficial.

This invention is directed toward a novel approach which substantiallyimproves steps 1-4 as given above. The method can also be used to createand optimize compounds that are substantially more effective and lesstoxic than typical experimental drugs that have been identified,optimized or profiled using standard, less sophisticated approaches thatare currently in use.

The methodology described herein has been developed as part of anintensive effort to develop advanced new pharmaceutical technologiesthat convert the “drug discovery” process into one more accuratelydescribed as a “drug creation” process by inventing predictable,reliable methodologies that provide the skilled investigator with thenecessary tools to create new drugs that target specific proteins ofimportance in human disease while reducing the time and immense costsassociated with the drug discovery/development process.

The progressive development of drug resistance in a patient is thehallmark of chronic treatment with many classes of drugs, especially inthe therapeutic areas of cancer and infectious diseases. Molecularmechanisms have been identified which mediate certain types of drugresistance phenomena, whereas in other cases the mechanisms of acquiredas well as de novo resistance remain unknown today.

One mechanism of induced (acquired) drug resistance originally thoughtto be relevant in the area of cancer therapy involves increasedexpression of a protein known as P-glycoprotein (P-gp). P-gp is locatedin the cell membrane and functions as a drug efflux pump. The protein iscapable of pumping toxic chemical agents, including many classicalanti-cancer drugs, out of the cell. Consequently, upregulation ofP-glycoprotein usually results in resistance to multiple drugs.Upregulation of P-glycoprotein in tumor cells may represent a defensemechanism which has evolved in mammalian cells to prevent damage fromtoxic chemical agents. Other related drug resistance proteins have nowbeen identified with similar functions to P-gp, includingmultidrug-resistance-associated protein family members such as MRP1 andABCG2. In any event, with the advent of the development of compoundsthat are specific for a given target protein, and less toxic, theimportance of P-glycoprotein and related ATP-binding cassette (ABC)transporter proteins in clinically significant drug resistance haslessened.

Another possible molecular mechanism of acquired drug resistance is thatalternative signal pathways are responsible for continued survival andmetabolism of cells, even though the original drug is still effectiveagainst its target. Furthermore, alterations in intracellular metabolismof the drug can lead to loss of therapeutic efficacy as well. Inaddition, changes in gene expression as well as gene amplificationevents can occur, resulting in increased or decreased expression of agiven target protein and frequently requiring increasing dosages of thedrug to maintain the same effects. (Adcock and Lane, 2003)

Mutation induced drug resistance is a frequently occurring event in theinfectious disease area. For example, several drugs have been developedthat inhibit either the viral reverse transcriptase or the viralprotease encoded in the human immunodeficiency (HIV) viral genome. It iswell established in the literature that repeated treatment ofHIV-infected AIDS patients using, for example, a reverse transcriptaseinhibitor eventually gives rise to mutant forms of the virus that havereduced sensitivity to the drug. Mutations that have arisen in the geneencoding reverse transcriptase render the mutant form of the enzyme lessaffected by the drug.

The appearance of drug resistance during the course of HIV treatment isnot surprising considering the rate at which errors are introduced intothe HIV genome. The HIV reverse transcriptase enzyme is known to beparticularly error prone, with a forward mutation rate of about 3.4×10⁻⁵mutations per base pair per replication cycle (Mansky et al., J. Virol.69:5087-94 (1995)). However, analogous mutation rates for endogenousgenes encoded in mammalian cells are more than an order of magnitudelower.

New evidence shows that drug resistance can also arise from a mutationalevent involving the gene encoding the drug target (Gorre et al.,Science, 2001; PCT/US02/18729). In this case, exposure of the patient toa specific therapeutic substance such as a given cancer drug thattargets a specific protein-of-interest (POI, or “target” protein) may befollowed by the outgrowth of a group of cells harboring a mutationoccurring in the gene encoding the protein that is the target of thetherapeutic substance. Whether the outgrowth of this population of cellsresults from a small percentage of pre-existing cells in the patientwhich already harbor a mutation which gives rise to a drug-resistantPOI, or whether such mutations arise de novo during or followingexposure of the animal or human being to a therapeutic agent capable ofactivating or inhibiting said POI, is presently unknown. In either case,such mutation events may result in a mutated protein (defined below as atheramutein) which is less affected, or perhaps completely unaffected,by said therapeutic substance.

Chronic myelogenous leukemia (CML) is characterized by excessproliferation of myeloid progenitors that retain the capacity fordifferentiation during the stable or chronic phase of the disease.Multiple lines of evidence have established deregulation of the Abltyrosine kinase as the causative oncogene in certain forms of CML. Thederegulation is commonly associated with a chromosomal translocationknown as the Philadelphia chromosome (Ph), which results in expressionof a fusion protein comprised of the BCR gene product fused to theAbelson tyrosine kinase, thus forming p210^(Bcr-Abl) which has tyrosinekinase activity. A related fusion protein, termed p190^(Bcr-Abl), thatarises from a different breakpoint in the BCR gene, has been shown tooccur in patients with Philadelphia chromosome positive (Ph+) AcuteLymphoblastic Leukemia (ALL) (Melo, 1994; Ravandi et al., 1999).Transformation appears to result from activation of multiple signalpathways including those involving RAS, MYC, and JUN. Imatinib mesylate(“STI-571” or “Gleevec®”) is a 2-phenylamino pyrimidine that targets theATP binding site of the kinase domain of Abl (Druker et al, NEJM 2001,p. 1038). Subsequently it has also been found by other methods to be aninhibitor of platelet-derived growth factor (PDGF) 0 receptor, and theKit tyrosine kinase, the latter of which is involved in the developmentof gastrointestinal stromal tumors (see below).

Until recently, it had not been observed that during the course oftreatment with a specific inhibitor of a given endogenous cellularprotein that a mutation in its corresponding endogenous gene could leadto the expression of protein variants whose cellular functioning wasresistant to the inhibitor. Work by Charles Sawyers and colleagues(Gorre et al., Science 293:876-80 (2001); PCT/US02/18729) demonstratedfor the first time that treatment of a patient with a drug capable ofinhibiting the p210^(Bcr-Abl) tyrosine kinase (i.e., STI-571) could befollowed by the emergence of a clinically significant population ofcells within said patient harboring a mutation in the gene encoding thep210^(Bcr-Abl) cancer causing target protein which contains the Abelsontyrosine kinase domain. Various such mutations gave rise to mutant formsof p210^(Bcr-Abl) which were less responsive to Gleevec treatment thanwas the original cancer causing version. Notably, the mutations thatemerged conferred upon the mutant protein a relative resistance to theeffects of the protein kinase inhibitor drug, while maintaining acertain degree of the original substrate specificity of the mutantprotein kinase. Prior to the work of Gorre et al., it was generallybelieved by those skilled in the art that the types of resistance thatwould be observed in patients exposed to a compound which inhibited theAbelson protein kinase, such as STI-571, would have resulted from one ormore of the other mechanisms of drug resistance listed above, or by someother as yet unknown mechanism, but that in any event said resistancewould involve a target (protein or otherwise) which was distinct fromthe drug's target POI.

Accordingly, the ability to treat clinically relevant resistant mutantforms of proteins that are otherwise the targets of an existing therapywould be extremely useful. Such mutated proteins (theramuteins asdefined below) are beginning to be recognized and understood to beimportant targets in recurring cancers, and will become important inother diseases as well. There exists a need for therapeutic agents thatare active against such drug resistant variant forms of cellularproteins that may arise before, during or following normally effectivedrug therapies. A key purpose of this invention is to provide ageneralizable methodology that the skilled investigator may utilize toidentify hits from high throughput screening (HTS) systems, create andoptimize lead compounds, and profile the spectrum of biological activityof such compounds, all without reliance upon older methods such as cellfree radioligand binding assays and the like. An additional key purposeof this invention is to provide compounds that may serve as potentialtherapeutic agents useful in overcoming mutation-induced drug resistancein endogenously occurring proteins.

BRIEF SUMMARY OF THE INVENTION

The method described herein involves the generation of a cellularresponse-based drug discovery and creation system that utilizesmodulations of a defined, pre-determined characteristic of a cell termedaphenoresponse as a tool to measure the ability of a given compound(chemical agent, modulator) to activate or inhibit a selected targetprotein. Through the iterative application of this process, themethodology described herein may be utilized to identify proteinmodulators (as herein defined), perform lead optimization on suchmodulators, and biologically profile the target protein specificity andselectivity of such modulators.

The invention described herein may be utilized with any target proteinand any eukaryotic cell type, provided however that an essential elementof the invention which is termed the phenoresponse is first identifiedand utilized according to the teachings herein. One embodiment of themethod provides the skillled investigator with the ability to identifyinhibitors or activators of a selected target protein. Anotherembodiment allows the skilled investigator to do rapid lead optimizationstudies in order to arrive at a potential clinical candidate compound.Still another embodiment provides the skilled investigator with theability to design compounds possessing a desired degree of specificityfor a given target protein as well as selectivity for that proteinrelative to distinct yet closely related family members of the targetprotein that may exist with certain targets.

Improvement of the therapeutic efficacy of a compound, including analready approved medication, is an important recurring problem inpharmaceutical research. A commonly utilized approach is to start withknown chemical structure and make additional chemical modifications tothe structure for the purpose of improving its potency, specificity (forthe target protein), or other parameter relevant to its therapeuticefficacy in the patient. In some cases the starting structure may be aknown drug. In other instances it may simply be an initial screening hitidentified either using a cell-free or primary cell-based screeningassay. In still other instances, the compound may be an initial chemicalstructure defined in its minimal terms based upon a screening hit orother model structure, and frequently termed a “scaffold”. For thepurposes of this invention, a scaffold is defined as a chemicalstructure with one or more side chains or ring substituents that havebeen removed relative to a representative compound that otherwise sharesthe same scaffold. By way of example, the third compound in Table 4 maybe thought of as a scaffold.

An important contribution of the present invention is the use of thephenoresponse, taken together with determination of the cellularspecificity of a first compound relative to a second compound in orderto determine whether the first compound exhibits an improved cellularspecificity relative to the second compound. This approach, reported forthe first time in the invention described herein, represents afundamental advance over the prior art. The prior art relies uponcell-free assay systems utilizing purified or recombinantly producedproteins for assaying the activity of a compound, and compares theeffect of a given compound on a target protein with its effects on otherproteins generally related (closely or distantly) to the target protein.Numerous examples of this type of prior art approach are found in theliterature, including Hanke et. al., 1996, Warmuth et. al., US2003/0162222 A1, Knight and Shokat, 2005, and references therein. Sucholder types of cell-free approaches are markedly less effective orcompletely ineffective as compared to the present invention inidentifying and optimizing the cellular specificity and therapeuticefficacy of a given scaffold. The substantial improvement of the presentinvention results from at least three key elements.

First, the concept of the phenoresponse, when utilized together with themeasurement of the cellular specificity of a given compound (as measuredfor example by determination of its CSG), provides a system which allowsthe identification of compounds that may interact with the targetprotein in an improved, more functionally effective manner.

Second, the present invention provides a method of identifying compoundsthat are also capable of interacting with other cellular componentsdistinct from the target protein (which include but are not limited toupstream or downstream components of a signal transduction pathwayinvolving the target protein such as monomeric or multi-subunitproteins, protein complexes, protein/nucleic acid complexes, and thelike), that are functional in the specific signal tranduction pathwaysor peripheral to the signal transduction pathways in which the targetprotein functions within the cell, to promote the disease state ofinterest such as a selected form of human cancer. Due to the complexityof the signal transduction cascades present in the cells of higherordered organisms such as humans, the current state of the art isincapable of complete knowledge regarding all of the mechanism in whicha given target protein functions within the cell.

Third, the present invention eliminates compounds that cross react withother non-target proteins that do NOT participate in the signaltransduction pathways that underlie the disease state in which thetarget protein functions. This ability of the present invention toeliminate such compounds (which will have untoward side effects in thepatient) arises from the direct comparative measurement of the cellularspecificity of the compound using the phenoresponse, which inherentlyeliminates effects upon the control cell. If the effect of a given testcompound results in a reduced cellular specificity as compared to thereference compound, the compound can be eliminated immediately. Whetherthe test compound is less effective against the target protein, orcross-reacts with other non-target proteins that do not participate inthe signal tranduction pathways of the target protein that modulate thephenoresponse linked to the target protein, or is simply cytotoxic, isirrelevant and only of academic interest. The essential point is thatthe test compound will be a less effective therapeutic and can beeliminated from further consideration. This saves the skilledinvestigator time and effort in evaluating variant chemical structures.It is important for the reader to recognize that compounds that may bevery potent and highly effective against the target in cell-free assaysystems may nevertheless show relatively low CSG determinations and maytherefore be rapidly eliminated, saving time and precious resources.

The aforementioned key advantages of the present invention are nowhereto be found in the prior art, and provide the essential improvements ofthe present invention over the prior art. These advantages areapplicable to all potential therapeutic target proteins, but areespecially important in the case of the intractable, highly drugresistant target proteins known as theramuteins (WO 2005/115992).

As a result of the use of this invention, the problem of improving andoptimizing a given compound relative to other less effective compoundsis greatly simplified and enhanced. The skilled investigator simplybegins with a first compound, whether it be an approved drug, ascreening hit, or a basic scaffold which is known to inhibit or activatethe protein of interest, and uses this first compound as an startingpoint for reference purposes. Additional compounds that are analogs,homologs, isomers, and the like, of the first compound (also referred toherein as the “starting compound” or “reference compound”) are thensynthesized using basic methods of medicinal chemistry synthesis whichare now standard in the art. Some of these chemical synthesis methodshave already been referred to in other sections herein, and the readermay also refer to Burbaum et al., 1995 and Goodnow et al., 2003 asgeneral references for such procedures. Once the additional compoundsare synthesized, the skilled investigator then proceeds to use themethods of the invention rather than the prior art method of constantlyreferring to the results obtained with cell-free assays by testing thenew compounds on both the target protein and an array of othernon-target proteins in an attempt to minimize the cross-reactivity ofthe compound with other proteins. Instead, through the use of thisinvention, the skilled investigator may guide the improvement of thechemical structure of the starting compound through direct reference tothe results obtained from determinations of the CSG of each compound tobe tested using the phenoresponse-based cellular assay system of thepresent invention. Most importantly, continuous reliance upon theresults of cell-free, purified protein assays, including “kinase panels”as referenced above in Hanke et al. (1996) and Knight and Shokat (2005)is eliminated in its entirety, and yet the compounds that result fromthe implementation of the present method are superior to those obtainedby the older methods, as shown by the activities of the compoundsidentified herein that are effective against the highly drug-resistanttheramutein p210 Bcr-Abl T315I, as shown in Table 4. Nothing limits theskilled investigator to independently test any resulting compounds in acell-free system for independent verification if so desired, but this isin no way required in order to practice the invention.

Prior to this invention, it has not been demonstrated that a cellularresponse-based drug discovery system is capable of identifying and rankordering inhibitors or activators of a selected target protein withoutprior reference to a cell-free, purified protein ligand binding assay orenzyme assay (when the target protein is an enzyme) in order toestablish that the compounds under investigation are actually binding tothe target protein.

These results demonstrate, for the first time, the use of a cellularresponse-based assay system as a primary tool to identify inhibitors oractivators of a given target protein from compounds that scorepositively in a high-throughput screen (HTS). These results alsodemonstrate that once a hit or lead compound capable of activating orinhibiting a given target protein is identified (by any method,including the embodiments disclosed herein or via classical cell-freeHTS methods), said compound may also be chemically optimized (i.e. leadoptimization may be performed on said compound) entirely using thephenoresponse-based cellular assay system without subsequent dependenceupon a cell-free purified protein assay system to independentlyverify/confirm that the inhibitory or activating ability of eachsubsequent compound synthesized during the lead optimization process.This embodiment alone saves the skilled investigator a substantialamount of time, effort and significant laboratory resources that wouldnormally be spent on generating and independently confirming inhibitoryor activating properties using classical cell-free purified proteinassays, radioligand binding assays, and the like.

The method is demonstrated herein using a specific mutated form of acancer-causing protein involved in the development and progression ofchronic myelogenous leukemia (CML). This protein, termed the Abelsonprotein kinase, in its cancer causing form is a known target for certaintyrosine kinase inhibitors such as imatinib mesylate. However, asdiscussed in detail below, this target protein can arise in a patient ina mutated form that becomes resistant to the inhibitory effects ofimatinib. Such forms of the Abelson kinase are termed theramuteins. Inan embodiment of the invention, suitable lead compounds capable ofinhibiting or activating a given theramutein are identified. In anotherembodiment of this invention, a lead compound is optimized. The methodis effective for the identification of hits, for lead optimization ofsuch hits (regardless of how such hits were initially identified), andfor biological profiling of compounds directed towards non-theramuteinendogenous target proteins. The general utility of the method isdemonstrated using a theramutein consisting of a mutated form of theAbelson kinase harboring a T315I mutation that confers a high degree ofdrug resistance.

This invention further relates to agents that are inhibitors oractivators of variant forms of proteins. The invention also relates toagents that are inhibitors or activators of certain variant forms ofendogenous proteins. Of particular interest are inhibitors andactivators of endogenous protein variants, encoded by genes which havemutated, which variants often arise or are at least first identified ashaving arisen following exposure to a chemical agent which is known tobe an inhibitor or activator of the corresponding unmutated endogenousprotein. Such protein variants (mutant proteins) are herein termed“theramuteins,” and may occur either spontaneously in an organism (andbe pre-existing mutations in some cases), or said mutants may arise as aresult of selective pressure which results when the organism is treatedwith a given chemical agent capable of inhibiting the non-mutated formof said theramutein (herein termed a “prototheramutein”). It will beunderstood that in some cases a prototheramutein may be a “wild type”form of a POI (e.g., a protein that gives rise to a disease due todisregulation). In other cases, the prototheramutein will be a diseasecausing variant of a “wild type” protein, having already mutated andthereby contributing to the development of the diseased state as aresult of said prior mutation. One example of the latter type ofprototheramutein is the P210^(BCR-ABL) oncoprotein, and a mutant form ofthis protein harboring a threonine (T) to isoleucine (I) mutation atposition 315 is termed P210^(BCR-ABL-T315I) and is one example of atheramutein. As used herein, the designation “P210^(BCR-ABL)” issynonymous with the term “p210^(Bcr-Abl)”, the “wild-type Bcr-Ablprotein”, and the like.

Theramuteins are a rare class of endogenous proteins that harbormutations that render said proteins resistant to drugs that are known toinhibit or activate in a therapeutically effective manner theirnon-mutated counterparts. The endogenous genes encoding a few suchproteins are presently known to exhibit such mutations under certaincircumstances. In one embodiment, this invention is directed towardcompositions that inhibit certain drug-resistant mutants (theramuteins)of the Abelson tyrosine kinase protein, originally termed P210-Bcr-Ablin the literature, that is involved in the development of chronicmyelogenous leukemia.

The present method is particularly directed toward the identification ofspecific inhibitors or specific activators of proteins. Use of the term“specific” in the context of the terms “inhibitor” or “activator” (seedefinitions below) means that said inhibitor or activator binds to theprotein and inhibits or activates the cellular functioning of theprotein without also binding to and activating or inhibiting a widevariety of other proteins or non-protein targets in the cell. Theskilled investigator is well aware that there is a certain degree ofvariability in the medical literature with respect to the concept of aspecific inhibitor or a specific activator, and of the related conceptof target protein “specificity” when discussing the actions ofinhibitors or activators of a protein. Accordingly, for the purposes ofthis invention, a substance is a specific inhibitor or a specificactivator of a given protein if said substance is capable of inhibitingor activating said protein at a given concentration such that acorresponding phenoresponse is modulated in the appropriate manner,without having an appreciable effect at the same given concentrationupon the phenoresponse (if any) of a corresponding control cell thatessentially does not express either the protein.

In certain embodiments, a substance may be a modulator of two closelyrelated proteins such as a prototheramutein and one of its correspondingtheramuteins. In other embodiments, in addition to being a modulator ofthe prototheramutein and theramutein, a substance may also modulate theactivities of proteins that have similar functions. As discussed above,in addition to inhibiting the p210^(Bcr-Abl) tyrosine kinase, imatinibmesylate is also capable of inhibiting the c-kit oncogene product (alsoa tyrosine kinase) which is overexpressed in certain gastrointestinalstromal tumors, as well as the PDGF β receptor (also a tyrosine kinase),which is expressed in certain chronic myelomonocytic leukemias (CMML).Such a compound is sometimes termed a “moderately specific” inhibitor.

The invention also provides a general method that can be used toidentify substances that will activate or inhibit a theramutein, to thesame extent, and preferably to an even greater extent than a known drugsubstance is capable of inhibiting the corresponding “wild type” form ofthat protein. (The skilled artisan is well aware, however, that said“wild type” forms of such proteins may have already mutated in thecourse of giving rise to the corresponding disease in which said proteinparticipates.)

In a preferred embodiment, the present invention provides inhibitors ofthe P210^(BCR-ABL-T315I) theramutein having the formula I

wherein:ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)(CH₂)_(q)C(O)R¹,    —(CH₂)_(p)N(R¹²)(R¹³), —(CH₂)_(p)N(R¹¹)(CH₂)_(q)R¹¹, —N(R¹¹)SO₂R¹¹,    —OC(O)N(R¹²)(R¹³), —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic    ring, and additionally or alternatively, two R¹ groups on adjacent    ring atoms form a 5- or 6-membered fused ring which contains from 0    to 3 heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom; wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰, CO₂R⁰,        C(O)R⁰, halo, aryl, and a heterocyclic ring;

p is 0 to 4;

q is 0 to 4;

R² is selected from —CR²¹ _(a)-, —NR²² _(b)-, and —(C═R²³)—;

each R²¹ is independently selected from H, halo, —NH₂, —N(H)(C₁₋₃alkyl),—N(C₁₋₃alkyl)₂,

—O—(C₁₋₃alkyl), OH and C₁₋₃ alkyl;

each R²² is independently selected from H and C₁₋₃ alkyl;

R²³ is selected from O, S, N—R⁰, and N—OR⁰;

R³ is selected from —CR³¹ _(c)-, —NR³² _(d)-, —SO₂—, and —(C═R³³)—;

-   -   each R³¹ group is selected from H, halo, —NH₂, —N(H)(R⁰),        —N(R⁰)₂, —O—R, OH and C₁₋₃ alkyl;    -   each R³² group is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, aralkyl, CO₂R⁰, C(O)R⁰, aryl, and a heterocyclic ring;    -   R³³ is selected from O, S, N—R³⁴, and N—OR⁰;    -   R³⁴ is selected from H, NO₂, CN, alkyl, cycloalkyl, alkenyl,        alkynyl, aralkyl, aryl and a heterocyclic ring;        R⁴ is selected from —CR⁴¹ _(e)-, —NR₄₂ ^(f)-, —(C═R⁴³)—, —SO₂—,        and —O—; each R⁴¹ is selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, CO₂R⁰, C(O)R⁰, aralkyl, aryl, and a        heterocyclic ring;    -   each R⁴² group is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, aralkyl, CO₂R⁰, C(O)R⁰, aryl, and a heterocyclic ring;    -   each R⁴³ is selected from O, S, N—R⁰, and N—OR⁰;        with the provisos that when R² is —NR²² _(b)- and R⁴ is —NR⁴²        _(f)-, then R³ is not —NR³² _(d)-; that both R³ and R⁴ are not        simultaneously selected from —(C═R³³)— and —(C═R⁴³)—,        respectively; and that R³ and R⁴ are not simultaneously selected        from —SO₂—;        R⁵ is selected from —Y—R⁶ and —Z—R⁷;

Y is selected from a chemical bond, O, NR⁰,

-   -   R⁶ is selected from alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   Z is a hydrocarbon chain having from 1 to 4 carbon atoms, and        optionally substituted with one or more of halo, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CO₂R⁰, C(O)R⁰,        C(O)N(R⁰)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁰;

R⁷ is H or is selected from aryl and a heterocyclic ring;

-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring;    a is 1 or 2;    b is 0 or 1;    c is 1 or 2;    d is 0 or 1;    e is 1 or 2; and    f is 0 or 1.

The invention provides for a fundamentally new way of treating cancerand other diseases where treatment with an existing drug compound, bywhatever mechanism, is followed by identifiable (clinically significant)theramutein-mediated drug resistance, by providing alternative drugsthat can be administered as theramuteins arise and are identified assuch (Wakai et al., 2004, reports an example wherein a theramutein mayarise during the course of an on-going treatment regimen), orpreemptively before the outgrowth of clinically significant populationsof theramutein expressing cells. Further, where a drug treatment for aparticular disease is less effective in a subset of individuals thatexpress a certain theramutein of a protein that the drug targets, theinvention enables the tailoring of treatments for those subjects byproviding alternative drug substances that will be effective againstsaid theramutein.

The invention provides a method of determining whether a chemical agentis at least as effective a modulator of a theramutein in a cell as aknown substance is a modulator of a corresponding prototheramutein. Oneembodiment of the method involves contacting a control cell thatexpresses the prototheramutein and is capable of exhibiting a responsivephenotypic characteristic (linked to the functioning of theprototheramutein in the cell) with the known modulator of theprototheramutein, contacting a test cell that expresses the theramuteinand is also capable of exhibiting the responsive phenotypiccharacteristic (linked to the functioning of the theramutein in thecell) with the chemical agent, and comparing the response of the treatedtest cell with the response of the treated control cell; to determinethat the chemical agent is at least as effective a modulator of thetheramutein as the known substance is a modulator of theprototheramutein. In certain other embodiments, one type of control cellmay not express the prototheramutein at all. In other embodiments, thecontrol cell may express about the same amount of the prototheramuteinas the test cell expresses of the theramutein. In still otherembodiments, the control cell may be capable of exhibiting theresponsive phenotypic characteristic to about the same extent as thetest cell under certain conditions. In additional embodiments, the testcell may express a given protein, whereas the control cell expresseslittle or essentially none of the protein.

Proteins of the invention that are of particular interest are thoseinvolved in regulatory function, such as enzymes, protein kinases,tyrosine kinases, receptor tyrosine kinases, serine threonine proteinkinases, dual specificity protein kinases, proteases, matrixmetalloproteinases, phosphatases, cell cycle control proteins, dockingproteins such as the IRS family members, cell-surface receptors,G-proteins, ion channels, DNA- and RNA-binding proteins, polymerases,and the like. No limitation is intended on the type of theramutein orother protein that may be used in the invention. At the present time,three theramuteins are known: BCR-ABL, c-Kit, and EGFR.

Any responsive phenotypic characteristic that can be linked to thepresence of the protein (including, e.g., a theramutein orprototheramutein) in the cell can be employed for use in the method,including, for example, growth or culture properties, thephosphorylation state (or other modification) of a substrate of thetheramutein, and any type of transient characteristic of the cell, aswill be defined and discussed in detail.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect on growth and viability of differentconcentrations of Compound 2 (C2) for non-transformed vector controlBa/F3 cells (which are IL-3 dependent) as well as Ba/F3 cells expressingthe “wild type” p210^(Bcr-Abl) (designated p210^(Bcr-Abl-wt)), and Ba/F3cells expressing the p210^(Bcr-Abl-T315I) drug resistant mutant. Cellcounts and viability were determined on an automated cell counter asdiscussed in detail in the specification. Cell counts are shown by thesolid color bars; cell viability is shown by the hashed bars. Note thatSTI-571 potently inhibits growth of the P210 cell line (grey bar)whereas it is unable to inhibit the growth of the T315I cell line (whitebar) even at 10 μM concentration. 500 nM C2 shows the largestspecificity gap within this dose-response series. Compare STI-571 at 10μM to C2 at 500 nM on the T315I cell line (white bars). Abbreviations:DMSO: dimethylsulfoxide (solvent used for drug dissolution).

FIG. 2 shows the effect on growth and viability of differentconcentrations of Compound 6 (C6) for non-transformed vector controlBa/F3 cells as well as Ba/F3 cells expressing the p210^(Bcr-Abl-T315I)drug resistant mutant. All other details are as per FIG. 1.

FIG. 3 shows various determinations of the specificity gap by comparingthe effects of various compounds identified in the screen in terms oftheir effects on the prototheramutein- and theramutein-expressing celllines. Compound 3 (C3) shows the best example of the ability of themethod to identify a compound that exerts an even greater effect on thetheramutein than on its corresponding prototheramutein. (Panel E). PanelA: control DMSO treatments; B: negative heterologous specificity gap; C:slightly positive heterologous specificity gap; D: large positivehomologous specificity gap; E: positive heterologous specificity gap.See text for explanations.

FIG. 4 shows an autoradiograph of recombinant P210 Bcr-Abl wild type andT315I mutant kinase domains assayed for autophosphorylation activity.200 ng of protein were preincubated with test substances for 10 minutesunder standard autophosphoryation reaction conditions and thenradiolabelled ATP was added and the reactions proceeded for 30 minutesat 30° C., after which the samples were separated by SDS-PAGE. The gelswere silver-stained, dried down under vacuum and exposed to X-ray film.Note that whereas 10 μM STI 571 is effective against wild type P210Bcr-Abl, it is virtually ineffective against the T315I kinase domain,even at concentrations up to 10 μM. “P210 cell line” refers to cellsexpressing p210^(BCR-ABL-wt). “T315I cell line” refers to cellsexpressing p210^(BCR-ABL-T315I).

FIG. 5 shows the chemical structures of representative compounds of thepresent invention.

FIG. 6 shows the chemical structures of representative compounds of thepresent invention.

FIG. 7 shows the chemical structures of representative compounds of thepresent invention.

FIG. 8 shows the chemical structures of representative compounds of thepresent invention.

FIG. 9 shows the chemical structures of representative compounds of thepresent invention.

FIG. 10 shows the chemical structures of representative compounds of thepresent invention.

FIG. 11 shows the chemical structures of representative compounds of thepresent invention.

FIG. 12 shows the chemical structures of representative compounds of thepresent invention.

FIG. 13 shows the chemical structures of representative compounds of thepresent invention.

FIG. 14 shows the inhibitory effect on growth rate of a hypotheticalcompound having a cellular specificity gap of 1 with respect to a testcell and a control cell.

FIG. 15 shows the inhibitory effect on growth rate of a hypotheticalcompound having a cellular specificity gap of 40 with respect to a testcell and a control cell.

FIG. 16 shows the growth inhibitory effect of imatinib mesylate atconcentrations significantly below the apparent IC₅₀ for cellulartoxicity.

FIG. 17 shows the effect on growth of different concentrations of C2 andvarious C2 analogues for Ba/F3 cells expressing the p210^(Bcr-Abl-T315I)drug resistant mutant.

FIG. 18 shows the results of a standard cell free protein kinaseautophosphorylation assay for T315I mutant kinase domains in thepresence of C2 and various C2 analogues at a concentration of 2 μM.

DETAILED DESCRIPTION OF THE INVENTION

The term “halo” or “halogen” as used herein includes fluorine, chlorine,bromine and iodine.

The term “alkyl” as used herein contemplates substituted andunsubstituted, straight and branched chain alkyl radicals having from 1to 6 carbon atoms. Preferred alkyl groups include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, and the like. Additionally, thealkyl group may be optionally substituted with one or more substituentsselected from halo, CN, CO₂R⁰, C(O)R, C(O)NR₂, NR₂, cyclic-amino, NO₂,and OR.

The term “cycloalkyl” as used herein contemplates substituted andunsubstituted cyclic alkyl radicals. Preferred cycloalkyl groups arethose with a single ring containing 3 to 7 carbon atoms and includecyclopropyl, cyclopentyl, cyclohexyl, and the like. Other cycloalkylgroups may be selected from C₇ to C₁₀ bicyclic systems or from C₉ to C₁₄tricyclic systems. Additionally, the cycloalkyl group may be optionallysubstituted with one or more substituents selected from halo, CN, CO₂R⁰,C(O)R, C(O)NR₂, NR₂, cyclic-amino, NO₂, and OR.

The term “alkenyl” as used herein contemplates substituted andunsubstituted, straight and branched chain alkene radicals. Preferredalkenyl groups are those containing two to six carbon atoms.Additionally, the alkenyl group may be optionally substituted with oneor more substituents selected from halo, CN, CO₂R⁰, C(O)R, C(O)NR₂, NR₂,cyclic-amino, NO₂, and OR.

The term “alkynyl” as used herein contemplates substituted andunsubstituted, straight and branched chain alkyne radicals. Preferredalkynyl groups are those containing two to six carbon atoms.Additionally, the alkynyl group may be optionally substituted with oneor more substituents selected from halo, CN, CO₂R⁰, C(O)R, C(O)NR₂, NR₂,cyclic-amino, NO₂, and OR.

The term “aralkyl” as used herein contemplates an alkyl group which hasas a substituent an aromatic group, which aromatic group may besubstituted and unsubstituted. The aralkyl group may be optionallysubstituted on the aryl with one or more substituents selected fromhalo, CN, CF₃, NR₂, cyclic-amino, NO₂, OR, CF₃, —(CH₂)_(x)R,—(CH₂)_(x)C(O)(CH₂)_(y)R, —(CH₂)_(x)C(O)N(R′)(R″),—(CH₂)_(x)C(O)O(CH₂)_(y)R, —(CH₂)_(x)N(R′)(R″), —N(R)SO₂R,—O(CH₂)_(x)C(O)N(R′)(R″), —SO₂N(R′)(R″), —(CH₂)_(x)N(R)—(CH₂)_(y)—R,—(CH₂)_(x)N(R)—C(O)—(CH₂)_(y)—R, —(CH₂)_(x)N(R)—C(O)—O—(CH₂)_(y)—R,—(CH₂)_(x)—C(O)—N(R)—(CH₂)_(y)—R, —(CH₂)C(O)N(R)—(CH₂)_(y)—R,—O—(CH₂)_(x)—C(O)—N(R)—(CH₂)_(y)—R, substituted and unsubstituted alkyl,substituted and unsubstituted cycloalkyl, substituted and unsubstitutedaralkyl, substituted and unsubstituted alkenyl, substituted andunsubstituted alkynyl, substituted and unsubstituted aryl, and asubstituted and unsubstituted heterocyclic ring, wherein the substitutedalkyl, substituted cycloalkyl, substituted aralkyl, substituted alkenyl,substituted alkynyl, substituted aryl, and substituted heterocyclic ringmay be substituted with one of more halo, CN, CF₃, CO₂R⁰, C(O)R,C(O)NR₂, NR₂, cyclic-amino, NO₂, and OR.

The term “heterocyclic group” or “heterocyclic ring” as used hereincontemplates aromatic and non-aromatic cyclic radicals having at leastone heteroatom as a ring member. Preferred heterocyclic groups are thosecontaining 5 or 6 ring atoms which includes at least one hetero atom,and includes cyclic amines such as morpholino, piperidino, pyrrolidino,and the like, and cyclic ethers, such as tetrahydrofuran,tetrahydropyran, and the like. Aromatic heterocyclic groups, also termed“heteroaryl” groups contemplates single-ring hetero-aromatic groups thatmay include from one to three heteroatoms, for example, pyrrole, furan,thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine,pyrazine, pyridazine, pyrimidine, and the like. The term heteroaryl alsoincludes polycyclic hetero-aromatic systems having two or more rings inwhich two atoms are common to two adjoining rings (the rings are“fused”) wherein at least one of the rings is a heteroaryl, e.g., theother rings can be cycloalkyls, cycloalkenyls, aryl, heterocycles and/orheteroaryls. Examples of polycyclic heteroaromatic systems includequinoline, isoquinoline, tetrahydroisoquinoline, quinoxaline,quinaxoline, benzimidazole, benzofuran, purine, imidazopyridine,benzotriazole, and the like. Additionally, the heterocyclic groups maybe optionally substituted with halo, CN, CF₃, NR₂, cyclic-amino, NO₂,OR, CF₃, —(CH₂)_(x)C(O)(CH₂)_(y)R, —(CH₂)_(x)C(O)N(R′)(R″),—(CH₂)_(x)C(O)O(CH₂)_(y)R, —(CH₂)_(x)N(R′)(R″), —N(R)SO₂R,—O(CH₂)_(x)C(O)N(R′)(R″), —SO₂N(R′)(R″), —(CH₂)_(x)N(R)—(CH₂)_(y)—R,—(CH₂)_(x)N(R)—C(O)—(CH₂)_(y)—R, —(CH₂)_(x)N(R)—C(O)—O—(CH₂)_(y)—R,—(CH₂)_(x)—C(O)—N(R)—(CH₂)_(y)—R, —(CH₂)_(x)C(O)N(R)—(CH₂)_(y)—R,—O—(CH₂)_(x)—C(O)—N(R)—(CH₂)_(y)—R, substituted and unsubstituted alkyl,substituted and unsubstituted cycloalkyl, substituted and unsubstitutedaralkyl, substituted and unsubstituted alkenyl, substituted andunsubstituted alkynyl, substituted and unsubstituted aryl, and asubstituted and unsubstituted heterocyclic ring, wherein the substitutedalkyl, substituted cycloalkyl, substituted aralkyl, substituted alkenyl,substituted alkynyl, substituted aryl, and substituted heterocyclic ringmay be substituted with one of more halo, CN, CF₃, CO₂R, C(O)R, C(O)NR₂,NR₂, cyclic-amino, NO₂, and OR.

The term “cyclic-amino” as used herein contemplates aromatic andnon-aromatic cyclic radicals having at least one nitrogen as a ringmember. Preferred cyclic amino groups are those containing 5 or 6 ringatoms, which includes at least one nitrogen, and includes morpholino,piperidino, pyrrolidino, piperazino, imidazole, oxazole, thiazole,triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine and thelike. Additionally, the cyclic-amino may be optionally substituted withhalo, CN, CF₃, NR₂, NO₂, OR, CF₃, substituted and unsubstituted alkyl,substituted and unsubstituted cycloalkyl, substituted and unsubstitutedaralkyl, substituted and unsubstituted alkenyl, substituted andunsubstituted alkynyl, substituted and unsubstituted aryl, and asubstituted and unsubstituted heterocyclic ring, wherein the substitutedalkyl, substituted cycloalkyl, substituted aralkyl, substituted alkenyl,substituted alkynyl, substituted aryl, and substituted heterocyclic ringmay be substituted with one or more of halo, CN, CF₃, CO₂R⁰, C(O)R,C(O)NR₂, NR₂, NO₂, and OR.

The term “aryl” or “aromatic group” as used herein contemplatessingle-ring aromatic groups (for example, phenyl, pyridyl, pyrazole,etc.) and polycyclic ring systems (naphthyl, quinoline, etc.). Thepolycyclic rings may have two or more rings in which two atoms arecommon to two adjoining rings (the rings are “fused”) wherein at leastone of the rings is aromatic, e.g., the other rings can be cycloalkyls,cycloalkenyls, aryl, heterocycles and/or heteroaryls. Additionally, thearyl groups may be optionally substituted with one or more substituentsselected from halo, CN, CF₃, NR₂, cyclic-amino, NO₂, OR, CF₃,—(CH₂)_(x)C(O)(CH₂)_(y)R, —(CH₂)_(x)C(O)N(R′)(R″),—(CH₂)_(x)C(O)O(CH₂)_(y)R, —(CH₂)_(x)N(R′)(R″), —N(R)SO₂R,—O(CH₂)_(x)C(O)N(R′)(R″), —SO₂N(R′)(R″), —(CH₂)_(x)N(R)—(CH₂)_(y)—R,—(CH₂)_(x)N(R)—C(O)—(CH₂)_(y)—R, —(CH₂)_(x)N(R)—C(O)—O—(CH₂)_(y)—R,—(CH₂)_(x)—C(O)—N(R)—(CH₂)_(y)—R, —CH₂)_(x)C(O)N(R)—(CH₂)_(y)—R,—O—(CH₂)_(x)—C(O)—N(R)—(CH₂)_(y)—R, substituted and unsubstituted alkyl,substituted and unsubstituted cycloalkyl, substituted and unsubstitutedaralkyl, substituted and unsubstituted alkenyl, substituted andunsubstituted alkynyl, substituted and unsubstituted aryl, and asubstituted and unsubstituted heterocyclic ring, wherein the substitutedalkyl, substituted cycloalkyl, substituted aralkyl, substituted alkenyl,substituted alkynyl, substituted aryl, and substituted heterocyclic ringmay be substituted with one of more halo, CN, CF₃, CO₂R⁰, C(O)R,C(O)NR₂, NR₂, cyclic-amino, NO₂, and OR.

The term “heteroatom”, particularly as a ring heteroatom, refers to N,O, and S.

Each R is independently selected from H, substituted and unsubstitutedalkyl, substituted and unsubstituted cycloalkyl, substituted andunsubstituted aralkyl, substituted and unsubstituted aryl and asubstituted and unsubstituted heterocyclic ring, wherein the substitutedalkyl, substituted cycloalkyl, substituted aralkyl, substituted aryl andsubstituted heterocyclic ring may be substituted with one or more halo,CN, CF₃, OH, CO₂H, NO₂, C₁₋₆alkyl, —O—(C₁₋₆alkyl), —NH₂, —NH(C₁₋₆alkyl)and —N(C₁₋₆alkyl)₂. Each R′ and R″ are independently selected from H, orsubstituted and unsubstituted alkyl, substituted and unsubstitutedcycloalkyl, substituted and unsubstituted aralkyl, substituted andunsubstituted aryl and a substituted and unsubstituted heterocyclicring, wherein the substituted alkyl, substituted cycloalkyl, substitutedaralkyl, substituted aryl and substituted heterocyclic ring may besubstituted with one or more halo, CN, CF₃, OH, CO₂H, NO₂, C₁₋₆alkyl,—O—(C₁₋₆alkyl), —NH₂, —NH(C₁₋₆alkyl) and —N(C₁₋₆alkyl)₂; or R′ and R″may be taken together with the nitrogen to which they are attached forma 5- to 7-membered ring which may optionally contain up to three furtherheteroatoms, which heteroatoms may be substituted by C₁₋₆alkyl. Each xand each y are independently selected from 0 to 4.

In a preferred embodiment, the present invention provides inhibitors ofthe P210^(BCR-ABL-T315I) theramutein having the formula I

wherein:ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)(CH₂)_(q)C(O)R¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom; wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰, CO₂R⁰,        C(O)R⁰, halo, aryl, and a heterocyclic ring;

p is 0 to 4;

q is 0 to 4;

R² is selected from —CR²¹ _(a)-, —NR²² _(b)-, and —(C═R²³)—;

-   -   each R²¹ is independently selected from H, halo, —NH₂,        —N(H)(C₁₋₃alkyl), —N(C₁₋₃alkyl)₂, —O—(C₁₋₃alkyl), OH and C₁₋₃        alkyl;    -   each R²² is independently selected from H and C₁₋₃ alkyl;    -   R²³ is selected from O, S, N—R⁰, and N—OR⁰;        R³ is selected from —CR³¹—, —NR³² _(d)-, —SO₂—, and —(C═R³³)—;    -   each R³¹ group is selected from H, halo, —NH₂, —N(H)(R⁰),        —N(R⁰)₂, —O—R, OH and C₁₋₃ alkyl;    -   each R³² group is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, aralkyl, CO₂R⁰, C(O)R⁰, aryl, and a heterocyclic ring;

R³³ is selected from O, S, N—R³⁴, and N—OR⁰;

R³⁴ is selected from H, NO₂, CN, alkyl, cycloalkyl, alkenyl, alkynyl,aralkyl, aryl and a heterocyclic ring;

R⁴ is selected from —CR⁴¹ _(e)-, —NR⁴² _(f)-, —(C═R⁴³)—, —SO₂—, and —O—;

-   -   each R⁴¹ is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, CO₂R⁰, C(O)R⁰, aralkyl, aryl, and a heterocyclic ring;    -   each R⁴² group is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, aralkyl, CO₂R⁰, C(O)R⁰, aryl, and a heterocyclic ring;    -   each R⁴³ is selected from O, S, N—R⁰, and N—OR⁰;        with the provisos that when R² is —NR²² _(b)- and R⁴ is —NR⁴²⁻,        then R³ is not —NR³² _(d)-; that both R³ and R⁴ are not        simultaneously selected from —(C═R³³)— and —(C═R⁴³)—,        respectively; and that R³ and R⁴ are not simultaneously selected        from —SO₂—;        R⁵ is selected from —Y—R⁶ and —Z—R⁷;    -   Y is selected from a chemical bond, O, NR⁰,    -   R⁶ is selected from alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   Z is a hydrocarbon chain having from 1 to 4 carbon atoms, and        optionally substituted with one or more of halo, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CO₂R⁰, C(O)R⁰,        C(O)N(R⁰)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁰;    -   R⁷ is H or is selected from aryl and a heterocyclic ring;

-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring;    a is 1 or 2;    b is 0 or 1;    c is 1 or 2;    d is 0 or 1;    e is 1 or 2; and    f is 0 or 1.

An important component and conceptual teaching of the Inventiondescribed herein is that neither the R² nor the R³ positions of thecompounds of this invention are members of any aromatic or non-aromaticring structure. We have discovered that compounds having the R² and/orthe R³ positions as members of any aromatic or non-aromatic ringstructure do not effectively inhibit the T315I theramutein, whereas thecompounds of the invention that lack such a ring component at thesepositions, in addition to having other preferred chemical groups, arepotent inhibitors of the T315I theramutein.

In preferred embodiments of the invention, ring A is an aromatic ring.

In preferred embodiments of the invention, X¹ or X² is N. In anotherpreferred embodiment, both X¹ and X² are N. In particularly preferredembodiments of the invention Ring A is a pyridine ring or a pyrimidinering. In still further preferred embodiments, Ring A is selected fromthe structures provided below:

In preferred embodiments of the invention, R⁵ is a group having theformula

wherein:R⁶¹ is selected from aryl and a heterocyclic ring;Q is selected from a chemical bond or a group having the formula —O—,—(CH₂)_(i)-, —(CH₂)_(i)C(O)(CH₂)_(j)-, —(CH₂)_(i)—N(R⁶²)—(CH₂)_(j)-,—(CH₂)_(i)C(O)—N(R⁶²)—(CH₂)_(j)-, —(CH₂)_(i)C(O)O(CH₂)_(j)-,—(CH₂)_(i)N(R⁶²)C(O)—(CH₂)_(j)-, —(CH₂)_(i)OC(O)N(R⁶²)—(CH₂)_(j)-, and—O—(CH₂)_(i)—C(O)N(R⁶²)—(CH₂)_(j)-;R⁶² is selected from H, alkyl, aryl, and a heterocyclic ring;

-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring;    h is 0 to 4;    i is 0 to 4; and    j is 0 to 4.

In further preferred embodiments of the invention, R⁵ is a group havingthe formula

wherein:

X³ is N or CH;

Q¹ is selected from a chemical bond or a group having the formula —O—,—CH₂—, —NH—, —C(O)—NH—, —C(O)O—, —NH—C(O)—, —OC(O)NH—, and —O—C(O)NH—;each R⁷⁰ is selected from halo, alkyl, CN, N(R⁷¹)₂, cyclic-amino, NO₂,OR⁷¹, and CF₃,each R⁷¹ is selected from H, alkyl, aryl, aralkyl and a heterocyclicring; andk is 0 to 4.

In further preferred embodiments of the invention, R⁵ is a group havingthe formula

wherein

X³ is N or CH;

Q¹ is selected from a chemical bond or a group having the formula —O—,—CH₂—, —NH—, —C(O)—NH—, —C(O)O—, —NH—C(O)—, —OC(O)NH—, and —O—C(O)NH—;R⁷⁰ is selected from halo, alkyl, CN, N(R⁷¹)₂, cyclic-amino, NO₂, OR⁷¹,and CF₃; and each R⁷¹ is selected from H, alkyl, aryl, aralkyl and aheterocyclic ring.In particularly preferred embodiments one or more of the followingselections is made: Q¹ is —NH—; X³ is N; each R⁷¹ is independentlyselected from H, methyl, and ethyl, and preferably each R⁷¹ is methyl;and/or R⁷⁰ is selected from OH, OCH₃, halo, and CF₃.

In a preferred embodiment, if R² or R⁴ is selected to be —NR²² _(b)- or—NR⁴²—, respectively, then R³¹ is not selected from halo, —NH₂,—N(H)(R⁰), —N(R⁰)₂, —O—R, or OH.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaI_(a)

wherein:ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)(CH₂)_(q)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom; wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰, CO₂R⁰,        C(O)R⁰, halo, aryl, and a heterocyclic ring;

p is 0 to 4;

q is 0 to 4;

each R²² is independently selected from H and C₁₋₃ alkyl;R³ is selected from —CR³¹ _(c)-, —NR³² _(d)-, —SO₂—, and —(C═R³³)—;

-   -   each R³¹ group is selected from H, halo, —NH₂, —N(H)(R⁰),        —N(R⁰)₂, —O—R, OH and C₁-3 alkyl;    -   each R³² group is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, aralkyl, CO₂R⁰, C(O)R⁰, aryl, and a heterocyclic ring;    -   R³³ is selected from O, S, N—R³⁴, and N—OR⁰;    -   R³⁴ is selected from H, NO₂, CN, alkyl, cycloalkyl, alkenyl,        alkynyl, aralkyl, aryl and a heterocyclic ring;        R⁴ is selected from —CR⁴¹ _(e)-, —NR⁴² _(f)-, —(C═R⁴³)—, —SO₂—,        and —O—;    -   each R⁴¹ is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, CO₂R⁰, C(O)R⁰, aralkyl, aryl, and a heterocyclic ring;    -   each R⁴² group is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, aralkyl, CO₂R⁰, C(O)R⁰, aryl, and a heterocyclic ring;    -   each R⁴³ is selected from O, S, N—R⁰, and N—OR⁰;        with the provisos that when R⁴ is —NR⁴² _(f)- then R³ is not        —NR³² _(d)-; and that both R³ and R⁴ are not simultaneously        selected from —(C═R³³)— and —(C═R⁴³)—, respectively; and that R³        and R⁴ are not simultaneously selected from —SO₂—;        R⁵ is selected from —Y—R⁶ and —Z—R⁷;    -   Y is selected from a chemical bond, O, N—R⁰,    -   R⁶ is selected from alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   Z is a hydrocarbon chain having from 1 to 4 carbon atoms, and        optionally substituted with one or more of halo, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CO₂R⁰, C(O)R⁰,        C(O)N(R⁰)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁰;    -   R⁷ is H or is selected from aryl and a heterocyclic ring;

-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring;    a is 1 or 2;    b is 0 or 1;    c is 1 or 2;    d is 0 or 1;    e is 1 or 2; and    f is 0 or 1.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaI_(b)

wherein:ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, (CH₂)_(p)N(R¹¹)_(q)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom; wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰, CO₂R⁰,        C(O)R⁰, halo, aryl, and a heterocyclic ring;

p is 0 to 4;

q is 0 to 4;

each R²² is independently selected from H and C₁₋₃ alkyl;

-   each R³² group is selected from H, alkyl, cycloalkyl, alkenyl,    alkynyl, aralkyl, CO₂R⁰, C(O)R⁰, aryl, and a heterocyclic ring;    R⁴ is selected from —CR⁴¹ _(e)-, —(C═R⁴³)—, —SO₂—, and —O—;    -   each R⁴¹ is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, CO₂R⁰, C(O)R⁰, aralkyl, aryl, and a heterocyclic ring;    -   each R⁴³ is selected from O, S, N—R⁰, and N—OR⁰;        R⁵ is selected from —Y—R⁶ and —Z—R⁷;    -   Y is selected from a chemical bond, O, N—R⁰,    -   R⁶ is selected from alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   Z is a hydrocarbon chain having from 1 to 4 carbon atoms, and        optionally substituted with one or more of halo, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CO₂R⁰, C(O)R⁰,        C(O)N(R⁰)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁰;    -   R⁷ is H or is selected from aryl and a heterocyclic ring;-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring;    a is 1 or 2;    b is 0 or 1;    c is 1 or 2;    d is 0 or 1;    e is 1 or 2; and    f is 0 or 1.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaI_(c)

whereinring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹,    —(CH₂)_(p)N(R¹¹)(CH₂)C(O)R¹¹CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹,    —OC(O)N(R¹²)(R¹³), —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic    ring, and additionally or alternatively, two R¹ groups on adjacent    ring atoms form a 5- or 6-membered fused ring which contains from 0    to 3 heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom, wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰, CO₂R⁰,        C(O)R⁰, halo, aryl, and a heterocyclic ring;

p is 0 to 4;

q is 0 to 4;

X³ is N, CH or C—R²;

-   each R² is independently selected from the group consisting of    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR²¹,    —(CH₂)_(r)C(O)(CH₂)_(s)R²¹, —(CH₂)_(r)C(O)N(R²²)(R²³),    —(CH₂)_(r)C(O)O(CH₂)_(s)R²¹, —(CH₂)_(r)N(R²¹)C(O)R²¹,    —(CH₂)_(r)N(R²²)(R²³), —N(R²¹)SO₂R²¹, —OC(O)N(R²²)(R²³),    —SO₂N(R²²)(R²³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R² groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    -   R²¹ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   R²² and R²³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R²² and R²³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom, wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰, CO₂R⁰,        C(O)R⁰, halo, aryl, and a heterocyclic ring;

r is 0 to 4;

s is 0 to 4;

m is 0 to 4;

-   R⁴ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl,    CO₂R⁰, C(O)R⁰, aryl, and a heterocyclic ring;    a is 0 or 1;    X⁴ is selected from

-   each R³ is independently selected from the group consisting of H,    —N(R⁰)₂, alkyl, cycloalkyl, alkenyl, alkynyl, CO₂R⁰, C(O)R⁰,    aralkyl, aryl, and a heterocyclic ring,-   R^(3′) is selected from H, —N(R⁰)₂, alkyl, cycloalkyl, aralkyl, aryl    and a heterocyclic ring, and-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

In preferred embodiments of the invention, R², R³ and R⁴ of formula Iare selected to give the following chemical groups:

-   -   —N(R²²)—N═C(R⁴¹)—    -   —N(R²²)—N(R³²)—C(═O)—    -   —N(R²²)—N(R³²)—C(R⁴¹)(R⁴¹)—    -   —N(R²²)—C(R³¹)(R³¹)—C(R⁴¹)(R⁴¹)—    -   —N(R²²)—C(R³¹)(R³¹)—C(═O)—    -   —N═N—C(R⁴¹)(R⁴¹)—    -   —C(R²¹)═C≡C(R⁴¹)—    -   —C(R²¹)═C(R³¹)—C(═O)—    -   —C(R²¹)═C(R³¹)—C(R⁴¹)(R⁴¹)—    -   —C(R²¹)(R²¹)—C(R³¹)═C(R⁴¹)—    -   —C(R²¹)(R²¹)—C(R³¹)(R³¹)—C(═O)—    -   —C(R²¹)(R²¹)—C(R³¹)(R³¹)—C(R⁴¹)(R⁴¹)—    -   —C(R²¹)(R²¹)—N(R³²)—C(═O)—    -   —C(R²¹)(R²¹)—N(R³²)—C(R⁴¹)(R⁴¹)—    -   —N(R²²)—C(═O)—C(R⁴¹)(R⁴¹)—    -   —N(R²²)—C(═O)—N(R⁴¹)—    -   —N(R²²)—C(═O)—O—    -   —C(R²¹)(R²¹)—C(═O)—C(R⁴¹)(R⁴¹)    -   —C(R²¹)(R²¹)—C(═O)—N(R⁴²)—    -   —N(R²²)—C(═NR³⁴)—N(R⁴²)—    -   —C(═O)—N(R³²)—N(R⁴²).        Particularly preferred chemical groups for R², R³ and R⁴        include:    -   —N(R²²)—N═C(R⁴¹)—    -   —N(R²²)—N(R³²)—C(═O)—    -   —N(R²²)—C(R³¹)(R³¹)—C(R⁴¹)(R⁴¹)—    -   —N(R²²)—C(R³¹)(R³¹)—C(═O)—    -   —C(R²¹)(R²¹)—C(═O)—C(R⁴¹)(R⁴¹)    -   —C(R²¹)(R²¹)—C(═O)—N(R⁴²)—    -   —N(R²²)—C(═NR³⁴)—N(R⁴²)—    -   —C(═O)—N(R³²)—N(R⁴²).

In further preferred embodiment, R⁶ or R⁷ is an aryl group, which may beoptionally substituted. Particularly preferred aryl groups includesubstituted or unsubstituted phenyl and pyridyl. In additional oralternative embodiments, it is preferred that the substituents R²¹ andR²² are independently selected from groups which have small steric bulkand are preferably selected from H and CH₃, and more preferably are H.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formula II

whereinring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)(CH₂)_(q)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom; wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN,        -   CF₃, NO₂, OR⁰, CO₂R⁰, C(O)R⁰, halo, aryl, and a heterocyclic            ring;

p is 0 to 4;

q is 0 to 4;

-   R⁸ is selected from the group consisting of is selected from H,    alkyl, cycloalkyl, alkenyl, alkynyl, CO₂R⁰, C(O)R⁰, aralkyl, aryl,    and a heterocyclic ring;-   R⁹ is selected from —Y—R⁶ and —Z—R⁷;

Y is selected from a chemical bond, O, N—R⁰,

-   -   R⁶ is selected from alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   Z is a hydrocarbon chain having from 1 to 4 carbon atoms, and        optionally substituted with one or more of halo, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CO₂R⁰, C(O)R⁰,        C(O)N(R⁰)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁰;    -   R⁷ is H or is selected from aryl and a heterocyclic ring; and

-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaII_(a)

whereinring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom, wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰, CO₂R⁰,        C(O)R⁰, halo, aryl, and a heterocyclic ring;

p is 0 to 4;

q is 0 to 4;

-   R⁸ is selected from the group consisting of is selected from H,    alkyl, cycloalkyl, alkenyl, alkynyl, CO₂R⁰, C(O)R⁰, aralkyl, aryl,    and a heterocyclic ring;-   X³ is N, CH or C—R⁵⁰;-   each R⁵⁰ is independently selected from the group consisting of    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁵¹,    —(CH₂)_(r)C(O)(CH₂)_(s)R⁵¹, —(CH₂)_(r)C(O)N(R⁵²)(R⁵³),    —(CH₂)_(r)C(O)O(CH₂)_(s)R⁵¹, —(CH₂)_(r)N(R⁵¹)C(O)R¹,    —(CH₂)_(r)N(R⁵²)(R⁵³), —N(R⁵¹)SO₂R⁵¹, —OC(O)N(R⁵²)(R⁵³),    —SO₂N(R⁵²)(R⁵³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R⁵⁰ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    -   R⁵¹ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   R⁵² and R⁵³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R⁵² and R⁵³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom, wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰, CO₂R⁰,        C(O)R⁰, halo, aryl, and a heterocyclic ring;

r is 0 to 4;

s is 0 to 4;

m is 0 to 4; and

-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaII_(b)

wherein:

-   R¹⁴ is selected from H and F;-   R⁸ is selected from the group consisting of is selected from H,    alkyl, cycloalkyl, alkenyl, alkynyl, CO₂R⁰, C(O)R⁰, aralkyl, aryl,    and a heterocyclic ring;-   X³ is N, CH or C—R⁶⁰;-   each R⁶⁰ is independently selected from the group consisting of    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰,    halo, aryl, and a heterocyclic ring;    R⁶¹ is selected from aryl and a heterocyclic ring;    Q is selected from a chemical bond or a group having the formula    —O—, —(CH₂)_(i)-, —(CH₂)_(i)C(O)(CH₂)_(j)-,    —(CH₂)_(i)—N(R⁶²)—(CH₂)—, —(CH₂)_(i)C(O)—N(R⁶²)—(CH₂)_(j)-,    —(CH₂)_(i)C(O)O(CH₂)_(j)-, —(CH₂)_(i)N(R⁶²)C(O)—(CH₂)_(j)-,    —(CH₂)_(i)OC(O)N(R⁶²)—(CH₂)_(j)-, and    —O—(CH₂)_(i)—C(O)N(R⁶²)—(CH₂)_(j)-;    R⁶² is selected from H, alkyl, aryl, and a heterocyclic ring;-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring;    h is 0 to 4;    i is 0 to 4; and    j is 0 to 4.

In preferred embodiments of compounds of the formula II_(b), R⁶⁰ isselected from halo, CF₃, and OH. In other preferred embodiments, R⁸ isselected from H and CH₃.

In preferred embodiments of compounds of the formula II_(b), X³ is N. Infurther preferred embodiments, Q is selected to be—(CH₂)_(i)—N(R⁶²)—(CH₂)_(j)-, and particularly in preferred embodiments,Q is —N(R⁶²)—.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaII,

wherein:ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom, wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰, CO₂R⁰,        C(O)R⁰, halo, aryl, and a heterocyclic ring;

p is 0 to 4;

q is 0 to 4;

R⁸ is selected from H and methyl;

X³ is N or CH;

R⁶¹ is selected from aryl and a heterocyclic ring;Q is selected from a chemical bond or a group having the formula —O—,—(CH₂)_(i)-, —(CH₂)_(i)C(O)(CH₂)_(j)-, —(CH₂)_(i)—N(R⁶²)—(CH₂)—,—(CH₂)_(i)C(O)—N(R⁶²)—(CH₂)_(j)-, —(CH₂)_(i)C(O)O(CH₂)_(j)-,—(CH₂)_(i)N(R⁶²)C(O)—(CH₂)_(j)-, —(CH₂)_(i)OC(O)N(R⁶²)—(CH₂)_(j)-, and—O—(CH₂)_(i)—C(O)N(R⁶²)—(CH₂)_(j)-;R⁶² is selected from H, alkyl, aryl, and a heterocyclic ring;

-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring;    h is 0 to 4;    i is 0 to 4; and    j is 0 to 4.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaII_(d)

wherein:ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR,    —(CH₂)_(p)C(O)(CH₂)_(q)R, —(CH₂)_(p)C(O)N(R¹²)(R³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹)C(O)R¹,    —(CH₂)_(p)N(R¹²)(R³), —N(R)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom, wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰, CO₂R⁰,        C(O)R⁰, halo, aryl, and a heterocyclic ring;

p is 0 to 4;

q is 0 to 4;

each R⁰ is independently selected from H, alkyl, cycloalkyl, aralkyl,aryl and a heterocyclic ring;R⁸ is selected from H and CH₃;

X³ is N or CH;

Q¹ is selected from a chemical bond or a group having the formula —O—,—CH₂—, —NH—, —C(O)—NH—, —C(O)O—, —NH—C(O)—, —OC(O)NH—, and —O—C(O)NH—;each R⁷⁰ is selected from halo, alkyl, CN, N(R⁷¹)₂, cyclic-amino, NO₂,OR⁷¹, and CF₃, each R⁷¹ is selected from H, alkyl, aryl, aralkyl and aheterocyclic ring; andk is 0 to 4.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaII_(e)

wherein:ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom, wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰, CO₂R⁰,        C(O)R⁰, halo, aryl, and a heterocyclic ring;

p is 0 to 4;

q is 0 to 4;

each R⁰ is independently selected from H, alkyl, cycloalkyl, aralkyl,aryl and a heterocyclic ring;R⁸ is selected from H and CH₃;

X³ is N or CH;

Q¹ is selected from a chemical bond or a group having the formula —O—,—CH₂—, —NH—, —C(O)—NH—, —C(O)O—, —NH—C(O)—, —OC(O)NH—, and —O—C(O)NH—;each R⁷⁰ is selected from halo, alkyl, CN, N(R⁷¹)₂, cyclic-amino, NO₂,OR⁷¹, and CF₃; and each R⁷¹ is selected from H and alkyl.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaII_(f)

whereinR⁸ is selected from H and CH₃;each R⁷⁰ is selected from halo, alkyl, CN, N(R⁷¹)₂, cyclic-amino, NO₂,OR⁷¹, and CF₃,each R⁷¹ is selected from H, alkyl, aryl, aralkyl and a heterocyclicring; andk is 0 to 4.

Exemplary compounds of the formula II, II_(a), II_(b), II_(c), II_(d),II_(e) or II_(f) includes the following structures:

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaIII

whereinring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom; wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰, CO₂R⁰,        C(O)R⁰, halo, aryl, and a heterocyclic ring;

p is 0 to 4;

q is 0 to 4;

R¹⁰ is selected from —Y′—R¹⁸;

-   Y′ is selected from a chemical bond, O, NR⁰—, and a hydrocarbon    chain having from 1 to 4 carbon atoms, and optionally substituted    with one or more of halo, alkyl, cycloalkyl, alkenyl, alkynyl,    aralkyl, CO₂R⁰, C(O)R⁰, C(O)N(R⁰)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁰;-   R¹⁸ is selected from the group consisting of H, alkyl, cycloalkyl,    alkenyl, alkynyl, aralkyl, CF₃, aryl, and a heterocyclic ring; and-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaIII_(a)

wherein:ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R³), —N(R¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom, wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰, CO₂R⁰,        C(O)R⁰, halo, aryl, and a heterocyclic ring;

p is 0 to 4;

q is 0 to 4;

X³ is N, CH or C—R⁵⁰;

-   each R⁵⁰ is independently selected from the group consisting of    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁵¹,    —(CH₂)_(r)C(O)(CH₂)_(s)R⁵¹, —(CH₂)_(r)C(O)N(R⁵²)(R⁵³),    —(CH₂)_(r)C(O)O(CH₂)_(s)R⁵¹, —(CH₂)_(r)N(R⁵¹)C(O)R⁵¹,    —(CH₂)_(r)N(R⁵²)(R⁵³), —N(R⁵¹)SO₂R⁵¹, —OC(O)N(R⁵²)(R⁵³),    —SO₂N(R⁵²)(R⁵³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R⁵⁰ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    -   R⁵¹ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   R⁵² and R⁵³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R⁵² and R⁵³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom, wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰, CO₂R⁰,        C(O)R⁰, halo, aryl, and a heterocyclic ring;

r is 0 to 4;

s is 0 to 4;

m is 0 to 4; and

-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315)1 theramutein having the formulaIII_(b)

wherein:ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom, wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰, CO₂R⁰,        C(O)R⁰, halo, aryl, and a heterocyclic ring;

p is 0 to 4;

q is 0 to 4;

X³ is N or CH;

R⁶¹ is selected from aryl and a heterocyclic ring;Q is selected from a chemical bond or a group having the formula —O—,—(CH₂)_(i)-, —(CH₂)_(i)C(O)(CH₂)_(j)-, —(CH₂)_(i)—N(R⁶²)—(CH₂)_(j)-,—(CH₂)_(i)C(O)—N(R⁶²)—(CH₂)_(j)-, —(CH₂)_(i)C(O)O(CH₂)_(j)-,—(CH₂)_(i)N(R⁶²)C(O)—(CH₂)_(j)-, —(CH₂)_(i)OC(O)N(R⁶²)—(CH₂)_(j)-, and—O—(CH₂)_(i)—C(O)N(R⁶²)—(CH₂)_(j)-;R⁶² is selected from H, alkyl, aryl, and a heterocyclic ring;

-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring;    h is 0 to 4;    i is 0 to 4; and    j is 0 to 4.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaIII_(c)

wherein:ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom, wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰, CO₂R⁰,        C(O)R⁰, halo, aryl, and a heterocyclic ring;

p is 0 to 4;

q is 0 to 4;

X³ is N or CH;

each R⁰ is independently selected from H, alkyl, cycloalkyl, aralkyl,aryl and a heterocyclic ring;Q¹ is selected from a chemical bond or a group having the formula —O—,—CH₂—, —NH—, —C(O)—NH—, —C(O)O—, —NH—C(O)—, —OC(O)NH—, and —O—C(O)NH—;each R⁷⁰ is selected from halo, alkyl, CN, N(R⁷¹)₂, cyclic-amino, NO₂,OR⁷¹, and CF₃,each R⁷¹ is selected from H, alkyl, aryl, aralkyl and a heterocyclicring; andk is 0 to 4.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T35)1 theramutein having the formulaIII_(d)

wherein:ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom, wherein the 5- to        7-membered ring may optionally be substituted with one to three        substituents that are independently selected from alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰, CO₂R⁰,        C(O)R⁰, halo, aryl, and a heterocyclic ring;

p is 0 to 4;

q is 0 to 4;

each R⁰ is independently selected from H, alkyl, cycloalkyl, aralkyl,aryl and a heterocyclic ring;R⁸ is selected from H and CH₃;

X³ is N or CH;

Q¹ is selected from a chemical bond or a group having the formula —O—,—CH₂—, —NH—, —C(O)—NH—, —C(O)O—, —NH—C(O)—, —OC(O)NH—, and —O—C(O)NH—;each R⁷⁰ is selected from halo, alkyl, CN, N(R⁷¹)₂, cyclic-amino, NO₂,OR⁷¹, and CF₃; andeach R⁷¹ is selected from H and alkyl.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaIII_(e)

whereinR¹⁴ is selected from H and F;each R⁷⁰ is selected from halo, alkyl, CN, N(R⁷¹)₂, cyclic-amino, NO₂,OR⁷¹, and CF₃,each R⁷¹ is selected from H, alkyl, aryl, aralkyl and a heterocyclicring; andk is 0 to 4.

Exemplary compounds of the formula III, III_(a), III_(b), III_(c),III_(d), or III_(e) includes the following structures:

In a further embodiment, the present invention provides inhibitors ofthe P210^(BCR-ABL-T315I) theramutein having the formula IV

wherein:ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R³), —N(R¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;

p is 0 to 4;

q is 0 to 4;

R²² is selected from H and C₁₋₃ alkyl;

-   R³⁴ is selected from H, NO₂, CN, alkyl, cycloalkyl, alkenyl,    alkynyl, aralkyl, aryl and a heterocyclic ring;-   R⁴⁴ is selected from H, alkyl, cycloalkyl, —(C═O)R⁰, alkenyl,    alkynyl, aralkyl, aryl, and a heterocyclic ring;-   R⁴⁵ is selected from —Y″—R¹⁹;-   Y″ is selected from a chemical bond, O, NR⁰—, and a hydrocarbon    chain having from 1 to 4 carbon atoms, and optionally substituted    with one or more of halo, alkyl, cycloalkyl, alkenyl, alkynyl,    aralkyl, CO₂R⁰, C(O)R⁰, C(O)N(R⁰)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁰;-   R¹⁹ is selected from the group consisting of H, alkyl, cycloalkyl,    alkenyl, alkynyl, aralkyl, CF₃, aryl, and a heterocyclic ring; and-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

Exemplary compounds of the formula IV include the following structures:

In a further embodiment, the present invention provides inhibitors ofthe P210^(BCR-ABL-T315I) theramutein having the formula V

wherein:ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂))C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R²)(R³, —N(R¹¹)SO₂R¹¹, —OC(O)N(R)(R¹³), —SO₂N(R¹²)(R¹³),    halo, aryl, and a heterocyclic ring, and additionally or    alternatively, two R¹ groups on adjacent ring atoms form a 5- or    6-membered fused ring which contains from 0 to 3 heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;

p is 0 to 4;

q is 0 to 4;

-   R²² is selected from H and C₁₋₃ alkyl;-   R³⁴ is selected from H, NO₂, CN, alkyl, cycloalkyl, alkenyl,    alkynyl, aralkyl, aryl and a heterocyclic ring;-   R⁵⁵ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl,    aralkyl, aryl, and a heterocyclic ring;-   R⁵⁶ is selected from —Y″—R¹⁹;-   Y″ is selected from a chemical bond, O, NR⁰—, and a hydrocarbon    chain having from 1 to 4 carbon atoms, and optionally substituted    with one or more of halo, alkyl, cycloalkyl, alkenyl, alkynyl,    aralkyl, CO₂R⁰, C(O)R⁰, C(O)N(R⁰)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁰;

R¹⁹ is selected from the group consisting of H, alkyl, cycloalkyl,alkenyl, alkynyl, aralkyl, CF₃, aryl, and a heterocyclic ring; and eachR⁰ is independently selected from H, alkyl, cycloalkyl, aralkyl, aryland a heterocyclic ring.

In a further embodiment, the present invention provides inhibitors ofthe P210^(BCR-ABL-T315I) theramutein having the formula V_(a)

wherein:ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;

p is 0 to 4;

q is 0 to 4;

-   R⁵⁵ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl,    aralkyl, aryl, and a heterocyclic ring;    X³ is N or C—R⁵⁰;-   each R⁵⁰ is independently selected from the group consisting of    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁵¹,    —(CH₂)_(r)C(O)(CH₂)_(s)R⁵¹, —(CH₂)_(r)C(O)N(R⁵²)(R⁵³),    —(CH₂)_(r)C(O)O(CH₂)_(s)R⁵¹, —(CH₂)_(r)N(R⁵¹)C(O)R⁵¹,    —(CH₂)_(r)N(R⁵²)(R⁵³), —N(R⁵¹)SO₂R⁵¹, —OC(O)N(R⁵²)(R⁵³),    —SO₂N(R⁵²)(R⁵³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R⁵⁰ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    -   R⁵¹ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   R⁵² and R⁵³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R⁵² and R⁵³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;

r is 0 to 4;

s is 0 to 4;

m is 0 to 4; and

-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

Exemplary compounds of the formula V or V_(a) include the followingstructures:

In a further embodiment, the present invention provides inhibitors ofthe P210^(BCR-ABL-T315I) or alternatively, two theramutein having theformula VI

wherein:ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂))C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R²)(R³), N(R¹¹)SO₂R¹¹, —OC(O)N(R)(R¹³), —SO₂N(R¹²)(R¹³),    halo, aryl, and a heterocyclic ring, and additionally or    alternatively, two R¹ groups on adjacent ring atoms form a 5- or    6-membered fused ring which contains from 0 to 3 heteroatoms;    n is 0 to 6, each R¹¹ is independently selected from H, alkyl,    cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic    ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;

p is 0 to 4;

q is 0 to 4;

R⁵⁵ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl,aryl, and a heterocyclic ring;

R⁵⁶ is selected from —Y″—R¹⁹;

-   Y″ is selected from a chemical bond, O, NR⁰—, and a hydrocarbon    chain having from 1 to 4 carbon atoms, and optionally substituted    with one or more of halo, alkyl, cycloalkyl, alkenyl, alkynyl,    aralkyl, CO₂R⁰, C(O)R⁰, C(O)N(R⁰)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁰;-   R¹⁹ is selected from the group consisting of H, alkyl, cycloalkyl,    alkenyl, alkynyl, aralkyl, CF₃, aryl, and a heterocyclic ring; and-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

In a further embodiment, the present invention provides inhibitors ofthe P210^(BCR-ABL-T315I) theramutein having the formula VI_(a)

wherein:ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;

p is 0 to 4;

q is 0 to 4;

-   R⁵⁵ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl,    aralkyl, aryl, and a heterocyclic ring;    X³ is N or C—R⁵⁰;-   each R⁵⁰ is independently selected from the group consisting of    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁵¹,    —(CH₂)_(r)C(O)(CH₂)_(s)R⁵¹, —(CH₂)_(r)C(O)N(R⁵²)(R⁵³),    —(CH₂)_(r)C(O)O(CH₂)_(s)R⁵¹, —(CH₂)_(r)N(R⁵¹)C(O)R¹,    —(CH₂)_(r)N(R⁵²)(R⁵³), —N(R⁵¹)SO₂R¹¹, —OC(O)N(R⁵²)(R⁵³),    —SO₂N(R⁵²)(R⁵³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R⁵⁰ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    -   R⁵¹ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   R⁵² and R⁵³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R⁵² and R⁵³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;

r is 0 to 4;

s is 0 to 4;

m is 0 to 4; and

-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

Exemplary compounds of the formula VI or VI_(a) include the followingstructures:

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaVII

wherein:ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fusedbicyclic ring;X¹ is selected from N, N—R⁰ or C—R¹;X² is selected from N, N—R⁰ or C—R¹;the dotted lines represent optional double bonds;

-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;

p is 0 to 4;

q is 0 to 4;

ring B is selected from a cycloalkyl group having 5 or 6 ring atoms, anda heterocyclic group containing 5 or 6 ring atoms which includes one tothree hetero atoms;

-   each R⁵⁰ is independently selected from the group consisting of    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁵¹,    —(CH₂)_(r)C(O)(CH₂)_(s)R⁵¹, —(CH₂)_(r)C(O)N(R⁵²)(R⁵³),    —(CH₂)_(r)C(O)O(CH₂)_(s)R⁵¹, —(CH₂)_(r)N(R⁵¹)C(O)R⁵¹,    —(CH₂)_(r)N(R⁵²)(R⁵³), —N(R⁵¹)SO₂R⁵¹, —OC(O)N(R⁵²)(R⁵³),    —SO₂N(R⁵²)(R⁵³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R⁵⁰ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    -   R⁵¹ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   R⁵² and R⁵³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R⁵² and R⁵³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;

r is 0 to 4;

s is 0 to 4;

m is 0 to 4; and

-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

Exemplary compounds of the formula VII include the following structures:

As used herein, the definition of each expression, e.g. alkyl, m, n, R,R′ etc., when it occurs more than once in any structure, is intended tobe independent of its definition elsewhere in the same structure.

For each of the above descriptions of compounds of the structures I,I_(a), I_(b), II, II_(a), etc., each recitation of the terms halo,alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, heterocyclic groupor heterocyclic ring, are independently selected from the definitions ofthese terms as provided in the beginning of this section.

It will be understood that chemical structures provided herein includethe implicit proviso that substitution is in accordance with permittedvalence of the substituted atom and the substituent(s), and that thesubstitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

When one or more chiral centers are present in the compounds of thepresent invention, the individual isomers and mixtures thereof (e.g.,racemates, etc.) are intended to be encompassed by the formulae depictedherein.

When one or more double bonds are present in the compounds of thepresent invention, both the cis- and trans-isomers are intended to beencompassed by the formulae depicted herein. Although chemicalstructures (such as, for example, structures II, IIa, V, V_(a), VI, andVI_(a)) are depicted herein in either cis of trans configuration, bothconfigurations are meant to be encompassed by the each of the formulae.

In certain embodiments, compounds of the invention may exist in severaltautomeric forms. Accordingly, the chemical structures depicted hereinencompass all possible tautomeric forms of the illustrated compounds.

The compounds of the invention may generally be prepared fromcommercially available starting materials and known chemical techniques.Embodiments of the invention may be synthesized as follows. One of skillin the art of medicinal or synthetic chemistry would be readily familiarwith the procedures and techniques necessary to accomplish the syntheticapproaches given below.

Compounds of the formula II may be prepared by reaction of anappropriate hydrazine compound, such as A, and an appropriate aldehyde,such as B, under conditions similar to those described on p. 562 ofGineinah, et al. (Arch. Pharm. Med. Chem. 2002, 11, 556-562).

For example, heating A with 1.1 equivalents of B for 1 to 24 hours in aprotic solvent such as a C₁ to C₆ alcohol, followed by cooling andcollection of the precipitate, would afford C. Alternatively, product Cmay be isolated by evaporation of the solvent and purification bychromatography using silica gel, alumina, or C₄ to C₁₈ reverse phasemedium. Similar methodology would be applicable in the cases where“Aryl” is replaced by other groups defined under R⁵.

Compounds of the formula III ring may be prepared by reaction of anappropriate hydrazine compound, such as D, and an activated carboxylicacid such as E, wherein LG is a leaving group such as halo,1-oxybenztriazole, pentafluorophenoxy, p-nitrophenoxy, or the like, orCompound E may also be a symmetrical carboxylic acid anhydride, wherebyconditions similar to those described on p. 408 of Nair and Mehta(Indian J. Chem. 1967 5, 403-408) may be used.

For example, treatment of D with an active ester such as Aryl-C(O)—OC₆F₅in an inert solvent such as dichloromethane, 1,2-dichloroethane, orN,N-dimethylformamide, optionally in the presence of a base such aspyridine or another tertiary amine, and optionally in the presence of acatalyst such as 4-N,N-dimethylaminopyridine, at an appropriatetemperature ranging from 0° C. to the boiling point of the solvent,would afford F, which may be isolated by evaporation of the solventfollowed by chromatography using silica gel, alumina, or C₄ to C₁₈reverse phase medium. The above active ester example of E would bereadily prepared from the corresponding carboxylic acid andpentafluorophenol using a carbodiimide such as dicyclohexylcarbodiimideas a condensing agent.

Precursors such as A and D may be prepared by reaction of an appropriatenucleophile, for example, a hydrazine derivative, with a heteroaromaticcompound bearing a halo substituent at a position adjacent to a nitrogenatom. For example, using methods analogous to those described by Wu, etal. (J. Heterocyclic Chem. 1990, 27, 1559-1563), Breshears, et al. (J.Am. Chem. Soc. 1959, 81, 3789-3792), or Gineinah, et al. (Arch. Pharm.Med. Chem. 2002, 11, 556-562), examples of compounds A and D may beprepared starting from, for example, a 2,4-dihalopyrimidine derivative,many of which are commercially available or are otherwise readilyprepared by one skilled in the art. Thus, treatment of an appropriate2,4-dihalopyrimidine derivative G with an amine or other nucleophile(Z), optionally in the presence of an added base, selectively displacesthe 4-halo substituent on the pyrimidine ring. Subsequent treatment ofthe product with a second nucleophilic reagent such as hydrazine or ahydrazine derivative, optionally in a solvent such as a C₁ to C₆ alcoholand optionally in the presence of an added base, displaces the 2-halosubstituent on the pyrimidine ring, to afford compounds that areexamples of structures A and D above.

Embodiments wherein R² is —NR²² and R³ is —C(═R³³) can be synthesized bymethods such as the following, or straightforward modifications thereof.The synthesis may be conducted starting from an appropriate ring Aderivative J that bears a leaving group (LG) adjacent to the requisitering nitrogen. Structure G above and the product of reaction ofstructure G with nucleophile Z, as illustrated above, are examples ofsuch appropriate Ring A derivatives J. Suitable LG′ groups are halo,alkylthio, alkylsulfonyl, alkylsulfonate or arylsulfonate. Treatment ofJ with an amine R¹²NH₂ effects displacement of LG′ to affordintermediates K. An example of this chemical transformation wherein R¹²is H and LG′ is CH₃SO₂— is reported by Capps, et al. J. Agric. FoodChem. 1993, 41, 2411-2415, and an example wherein R¹² is H and LG′ is Clis reported in Marshall, et al. J. Chem. Soc. 1951, 1004-1015.

Intermediates of structure K are transformed to compounds of theinvention by simultaneous or sequential introduction of the elements, ofR³, R⁴, and R⁵. For example, treatment of intermediates of structure Kwith individual isocyanates R⁶—N═C═O affords in a single step compoundsof structure M, which are compounds of the invention wherein R²═—NR²²—,R³═—C═O—, R⁴═—NH—, and R⁵=-chemical bond-R⁶. Alternative methods toconvert compounds of structure K to compounds of structure M are wellknown to those skilled in the art, wherein R³ together with a leavinggroup (for example p-nitrophenoxy or chloro) is first introduced,followed by subsequent displacement of the leaving group by, forexample, an amine R⁶—NH₂, to introduce R⁵ and R⁶.

Alternatively, treatment of intermediates of structure K with a reagentsuch as cyanamide (NH₂—CN), typically under conditions of heating andoptionally in the presence of acid in a solvent such as ethyl acetate ordioxane, affords intermediates N. Alternatives to cyanamide arenitroguanidine or amidinosulfonic acid (NH₂—C(═NH)—SO₃H). An example ofsuch a transformation using cyanamide is reported by Latham et al., J.Org. Chem. 1950, 15, 884. An example using nitroguanidine is reported byDavis, Proc. Natl. Acad. Sci. USA 1925, 11, 72. Use of amidinosulfonicacid was reported by Shearer, et al. Bioorg. Med. Chem. Lett. 1997, 7,1763.

In analogy to the conversion of intermediates A or D to embodimentsrepresented by C or F, intermediates K are converted, respectively, tocompounds represented by P or Q, which are further embodiments of theinvention.

Treatment of A or K with a ketone S, wherein R is as defined above, inplace of an aldehyde B in the schemes above, affords compounds ofstructure T or U, respectively, which are further embodiments of theinvention.

The non-guanidino carbon-nitrogen double bond of U can be selectivelyreduced by an appropriate reducing agent such as a metal (boron,aluminum, silicon, etc.) hydride reagents, preferably one with basicproperties, to afford compounds V of the invention.

Embodiments of the invention wherein R═CO, R═—NR³—, R═N—, and R⁵═ZR⁷,wherein Z is a hydrocarbon chain and R⁷ is as defined above, may beprepared as follows. When R³²═H, a Ring A-derived carboxylic acid W isactivated by conversion to the corresponding acid chloride, oralternatively to an active ester, or to an analogous activatedderivative, many of which are well known in the art. Treatment of theactivated carboxylic acid with hydrazine affords the correspondinghydrazide Y. Treatment of Y with an aldehyde or ketone (under conditionsof heating and/or mild acid catalysis if necessary) affords the desiredfinal product Z.

If not commercially available, Ring A-derived carboxylic acids W may beprepared by treatment of starting material J above with cyanide ion,optionally with heating or transition metal catalysis, to replace theleaving group LG′ with a cyano residue. Basic or acidic hydrolysis ofthe cyano group affords the desired carboxylic acid intermediate W.

When R³² is not H, then a protected form of monosubstituted hydrazinemay be used in the above scheme in place of hydrazine. Thus, treatmentof the activated carboxylic acid from W with R³²NHNH-PG, where PG is anitrogen protecting group such as benzyloxycarbonyl ort-butyloxycarbonyl, followed by deprotection and treatment with anappropriate aldehyde or ketone as above affords Z′, a further embodimentof the invention.

It will be apparent to a practitioner skilled in the art of organicmolecule synthesis that the reaction processes illustrated above arerepresentative of a broader set of methods that are logical extensionsof the illustrated processes. Thus, additional embodiments of theinvention that incorporate additional variants in R², R³, R⁴, and R⁵claimed by this invention are prepared by obvious modifications of theabove processes.

As would be recognized by a person of ordinary skill, it may beadvantageous to employ a temporary protecting group in achieving thefinal product. The phrase “protecting group” as used herein meanstemporary modifications of a potentially reactive functional group whichprotect it from undesired chemical transformations. Examples of suchprotecting groups include esters of carboxylic acids, silyl ethers ofalcohols, and acetals and ketals of aldehydes and ketones, respectively.The field of protecting group chemistry has been reviewed (Greene, T.W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2^(nd) ed.;Wiley: New York, 1991).

One embodiment of this invention is directed to any endogenouslyoccurring mammalian target protein selected by the skilled investigatorto be of interest for the identification and/or optimization of acompound as an inhibitor or activator of said protein. In general suchselected proteins will already be known to be involved in the etiologyor pathogenesis of a human disease. In another embodiment, the inventionis also directed toward mutant forms of such mammalian proteins. A“mutein” is a protein having an amino acid sequence that is altered as aresult of a mutation that has occurred in its corresponding gene (Weigelet al, 1989). Such mutations may result in changes in one or more of thecharacteristics of the encoded protein. For example, an enzyme variantthat has modified catalytic activity resulting from a change in one ormore amino acids is a mutein.

This invention is concerned with proteins harboring an alteration of atleast one amino acid residue (the terms “amino acid sequence change” or“amino acid sequence alteration” include changes, deletions, oradditions, of at least one amino acid residue, or any combination ofdeletions, additions, changes) such that the resulting mutein has become(as a result of the mutation) resistant to a known therapeutic agentrelative to the sensitivity of the non-mutated version of said proteinto the therapeutic agent. This specialized class of muteins ishereinafter referred to as a theramutein, and the corresponding proteinlacking the mutation is referred to herein as a prototheramutein.

As used herein, “prototheramutein” refers to an endogenously occurringprotein in a cell that is susceptible to mutation that confers relativeinsensitivity (i.e. resistance) to a therapeutic compound whichotherwise inhibits or activates the protein. Accordingly, “theramutein”refers to an endogenously occurring protein or portion of a protein in acell that contains at least one amino acid sequence alteration relativeto an endogenous form of the protein, wherein the amino acid sequencechange is or was identified or becomes identifiable, and is or has beenshown to be clinically significant for the development or progression ofa given disease, following exposure of at least one human being to asubstance that is known to inhibit or activate the prototheramutein.Solely for the purposes of defining the preceding sentence, a substanceneed not be limited to a chemical agent for the purposes of firstdefining the existence of a theramutein. Thus, by definition, atheramutein is a protein which harbors a mutation in its correspondingendogenous gene, wherein said mutation is associated with thedevelopment of clinical resistance in a patient to a drug that isnormally able to activate or inhibit the non-mutated protein. Withrespect to a given theramutein, the term “correspondingprototheramutein” refers to the prototheramutein which, throughmutation, gives rise to said theramutein. Similarly, with respect to agiven prototheramutein, the “corresponding theramutein” refers to thetheramutein which has arisen by mutation from said prototheramutein.

Accordingly, it is apparent to a skilled artisan that, as the geneswhich encode theramuteins are limited to endogenously occurring genes,the definition of a theramutein excludes proteins encoded bydisease-causing infectious agents such as viruses and bacteria. As usedherein, the term “endogenous gene” refers to a gene that has beenpresent in the chromosomes of the organism at least in its unmutatedform, since inception. The term “cell” as used herein refers to a livingeukaryotic cell whether in an organism or maintained under appropriatelaboratory tissue or organ culture conditions outside of an organism.

In one embodiment of the invention, the target protein (POI) may be anyendogenously encoded mammalian protein. In another aspect of theinvention, the POI is a theramutein, which is a protein that is alteredfor the first time with respect to a commonly occurring “wild type” formof the protein (i.e., a wild type protein is the prototheramutein fromwhich the theramutein arises). In yet another aspect of the invention, atheramutein is a variant of a protein that is, itself, already a mutein(i.e., a mutein is the prototheramutein from which the theramuteinarises). In still another embodiment, a theramutein may be furthermutated as compared to a previously existing theramutein. In suchinstances, the first theramutein (such as the T315I mutant of p210BCR-ABL (see below), may be thought of as a “primary” theramutein,whereas subsequent mutations of the (already mutated) T315I variant maybe termed a secondary theramutein, tertiary theramutein, etc. Asexemplified below, a mutein of the invention is a variant of Bcr-Abltyrosine kinase that escapes inhibition by an inhibitor of the “wildtype” Bcr-Abl. Such a Bcr-Abl mutein is altered with respect to a morecommon or “wild type” form of Bcr-Abl (which is also a mutein as well)in such a way that a property of the protein is altered.

It is understood that a protein of interest (POI) is an endogenouslyencoded mammalian protein. It will also be understood that a mutein ofprimary interest is a theramutein that may have the same, increased, ordecreased specific activity relative to its prototheramutein, and thatit is not inhibited or is poorly inhibited by an agent that is used toinhibit the prototheramutein. Likewise, another theramutein of primaryinterest is one that has the same, increased or decreased specificactivity (relative to its prototheramutein) and that is not activated oris poorly activated by an agent that is used to activate theprototheramutein. Other variations are obvious to the skilled artisan.It will be further appreciated that theramuteins can include naturallyoccurring or commonly observed variants of a protein, for example,variants that are expressed from different alleles of a particular gene.In some cases such variants may be unremarkable with respect to theirnormal cellular function, with functional differences becoming apparentonly in the presence of agents that differentially inhibit or activatethe cellular function of the variants. For example, naturally occurringvariants of a particular enzyme may have activity profiles that are notsubstantially different, but a therapeutic agent that modulates one maybe ineffective in modulating the other.

It will be appreciated that one aspect of the invention is theidentification of an agent that is active against a selected POI whosecellular function contributes to a given disease state such thatactivators or inhibitors of said POI would be expected to betherapeutically effective during the course of treatment for thedisease. No limitation of any kind or nature is intended on the type ofdisease that may be treated, nor on the type of protein that may betargeted for modulation according to the teachings herein, provided thatall other limitations stated herein are met, including the fact that anysuch protein that is selected for targeting must be an endogenousprotein. Obviously, the skilled investigator may use non-endogenouslyoccurring nucleic acids such as cDNAs in order to practice the methodtaught herein provided that the amino acid sequence corresponds to anendogenously occurring POI.

It will also be appreciated that, whereas one aspect of the invention isthe identification of an agent that is active against a protein ortheramutein that arises or becomes dominant (by any mechanism) prior toor during the course of a treatment for a given disease, another aspectis the identification of an agent that is active against a mutein thatis common within a population of unafflicted individuals, but whereinsaid mutein is less susceptible to modulation by an approved drug, andwhere the variation in the activity profile of the mutein becomesimportant (and is therefore first identified as being a theramutein) ina disease state such as where it is overexpressed or participates in asignaling process which has otherwise become abnormally regulated. Forexample, a neoplastic disease may be caused by abnormal regulation of acellular component other than the theramutein or its prototheramutein,and still be treatable with an inhibitor of the prototheramutein,whereas the same treatment would be less effective or ineffective wherethe theramutein was present. This can be an issue where it is observedthat the response of a particular tumor type to an anticancer agentvaries among individuals that express different variants of an enzymeagainst which the anticancer agent is directed (Lynch et al., 2004).Here, the variants would not have arisen or become predominant duringthe course of treatment of the disease, but are preexisting in thehealthy population and are detected only by their altered responsivenessto a particular course of established therapeutic treatment.

As used herein, the terms “agonist” and “activator” of a protein areused interchangeably. An activator (agonist) is limited to a substancethat binds to and activates the functioning of a given protein. Unlessexplicitly stated otherwise, an “activator”, an “agonist”, and an“activator of a protein” are identical in meaning. The activation by anactivator may be partial or complete. Likewise, as used herein, theterms “antagonist” and “inhibitor” of a protein are usedinterchangeably. An inhibitor (antagonist) is limited to a substancethat binds to and inhibits the functioning of a given protein. To statethat a substance “inhibit(s)” a protein means the substance binds to theprotein and reduce(s) the protein's activity in the cell withoutmaterially reducing the amount of the protein in the cell. Similarly, tostate that a substance “activate(s)” a protein, such as aprototheramutein or theramutein, is to state that the substanceincreases the defined function of the protein in the cell withoutsubstantially altering the level of the protein in the cell. Unlessexplicitly stated otherwise, an “inhibitor”, an “antagonist” and an“inhibitor of a protein” are also synonymous.

The inhibition by an inhibitor may be partial or complete. A modulatoris an activator or an inhibitor. By way of example, an “activator ofPKCp_(β1)” should be construed to mean a substance that binds to andactivates PKCp_(β1). Similarly, an “inhibitor of p210^(Bcr-Abl)” is asubstance that binds to and inhibits the functioning of p210^(Bcr-Abl).To state that a substance “inhibits a protein” requires that thesubstance bind to the protein in order to exert its inhibitory effect.Similarly, to state that a substance “activates protein X” is to statethat the substance binds to and activates protein X. The terms“bind(s),” “binding,” and “binds to” have their ordinary meanings in thefield of biochemistry in terms of describing the interaction between twosubstances (e.g., enzyme-substrate, protein-DNA, receptor-ligand, etc.).As used herein, the term “binds to” is synonymous with “interacts with”in the context of discussing the relationship between a substance andits corresponding target protein. As used herein, to state that asubstance “acts on” a protein, “affects” a protein, “exerts its effecton” a protein, etc., and all such related terms uniformly mean (as theskilled investigator is well aware) that said substance activates orinhibits said protein.

The concept of inhibition or activation of a mutated form of anendogenous protein to a greater extent than the correspondingnon-mutated counterpart protein is defined for the first time andreferred to herein as a positive “specificity gap.” In general terms,and using an inhibitor case as an example, the specificity gap refers tothe difference between the ability of a given substance, undercomparable conditions to inhibit the theramutein in a cell-based assaysystem of the invention as compared to either:

a) the ability of the same substance under comparable conditions toinhibit the prototheramutein; or

b) the ability of a second substance (usually a known inhibitor of theprototheramutein) to inhibit the theramutein under comparableconditions; or

c) the ability of the second substance to inhibit the prototheramuteinunder comparable conditions.

When the comparison is made between the effects of two distinctsubstances (tested individually) on the theramutein alone, the result istermed a homologous specificity gap determination.

Alternatively, when a comparison is made between the effects of twodistinct substances (generally, but not always), one of which is testedon the theramutein and the other on the prototheramutein, respectively,the result is termed a heterologous specificity gap (SG) determination.Thus, (a) and (c) as given above are examples of heterologousspecificity gap (SG) determinations (although (a) uses the samesubstance in both instances), whereas (b) is an example of a homologousspecificity gap determination.

Reference to FIG. 3 is informative in understanding and elucidatingthese concepts.

Analogous issues apply when the case concerns an activator. It will beimmediately obvious to the skilled artisan that the term “comparableconditions” includes testing two different compounds, for example, atthe same concentration (such as comparing two closely related compoundsto determine relative potency), or by comparing the effects of twodifferent compounds tested at their respective IC₅₀ values on thecorresponding prototheramutein and theramutein. The skilled investigatorwill easily recognize other useful variations and comparable conditions.

Thus, in one embodiment of the application of this approach, substancesthat are more effective against a theramutein have a “positivespecificity gap.” A “zero, null or no” specificity gap indicates thatthere is no significant measurable difference between the effect of asubstance on the theramutein as compared to its effect on theprototheramutein (however such compounds may be quite useful in theirability to inhibit or activate both a theramutein and its correspondingprototheramutein), and a “negative specificity gap” indicates asubstance that at a given concentration is less effective against thegiven theramutein than against a form of the correspondingprototheramutein or other comparative form of the theramutein (such asone that may harbor a different mutation). The latter category isgenerally of lesser interest than the former categories of compounds,except in the case where the compound is so potent that its relativelylesser effect on the theramutein is of no real concern from theperspective of therapeutic efficacy. The skilled investigator can easilyrecognize a variety of approaches to quantifying the specificity gapassessment in a manner tailored to his or her needs. Such an analysismay assist the skilled investigator in classifying various compoundsinto discrete categories that may be helpful in guiding further leadoptimization or biological profiling studies on such compounds.

The invention also provides a means for identifying compounds thatexhibit a desired specificity gap. Such compounds can be identified andtheir ability to inhibit or activate the theramutein determined using anin vitro cell-based assay system where the effect of a substance on thecellular functioning of the mutated endogenous form of the protein iscompared to the effect of the same drug on the cellular functioning of anon-mutated endogenous form of the protein.

Thus, the system enables the discovery of compounds capable of bindingto a theramutein and exerting a greater modulatory effect on thecellular functioning of said theramutein than on its correspondingprototheramutein. Further, the system enables the discovery of compoundscapable of binding to a theramutein and exerting at least as great orgreater modulatory effect on the cellular functioning of a theramuteinthan previously known compounds are able to exert on the correspondingprototheramutein. In a particular embodiment of the invention, acompound may be screened for and identified that 1) is at least aseffective against the theramutein as the original drug is against theprototheramutein, and/or 2) is similarly effective against theprototheramutein as against the theramutein (i.e., displays a small oressentially zero specificity gap).

In an embodiment of the invention, cells that overexpress a theramuteinof interest are used to identify chemical agents that are inhibitors oractivators of (i.e., that bind to and inhibit or that bind to andactivate) at least the selected theramutein. The chemical agents mayalso be inhibitors or activators of the prototheramutein or even othertheramuteins of the same prototheramutein. As used herein, the terms“chemical agent” and “compound” are used interchangeably, and both termsrefer exclusively to substances that have a molecular weight up to, butnot including, 2000 atomic mass units (Daltons). Such substances aresometimes referred to as “small molecules.” Unless otherwise statedherein, the term substance as used herein refers exclusively to chemicalagents/compounds, and does not refer to biological agents. As usedherein, “biological agents,” are molecules which include proteins,polypeptides, and nucleic acids, and have molecular weights equal to orgreater than 2000 atomic mass units (Daltons).

In one embodiment of the invention, a theramutein is selected and usedin a phenoresponse-based cellular assay system of the present inventiondesigned to identify agents that are inhibitors or activators of thetheramutein. Where two or more distinct theramuteins originating fromthe same prototheramutein are known, it is preferable to select the mostresistant theramutein available for use in the assay system. In general,the degree of resistance of a theramutein to a given chemical agent isdetermined relative to its non-mutated counterpart (prototheramutein)using the drug that was first administered and known to inhibit oractivate the prototheramutein and against which the theramutein “arose.”The methods of determining the degree of such resistance, for example byanalysis of IC₅₀ or AC₅₀ values, are well known and standard in the artand will not be reiterated herein. However, no causal relationship isnecessary or should be inferred between the treatment of the patientwith a given therapeutic agent per se and the subsequent appearance of atheramutein. Rather, what is required in order to practice the inventionas it pertains to theramuteins is that a true theramutein be properlyselected according to the teachings herein.

Thus, for example, randomly generated site directed mutants of knownproteins that are created in the laboratory but that have not been shownto be clinically relevant are not appropriate muteins for use within thescope of this invention. Such muteins would not, of course, be properlyclassified as theramuteins either.

For example, in an effort to obtain potential inhibitors of mutants ofp210^(Bcr-Abl), Huron et al. (2003) used a recombinant c-abl preparationand screened a series of compounds known to inhibit c-src tyrosinekinase activity. The authors performed c-abl kinase assays on theircompounds and identified the most potent compound as an 8 nM inhibitoragainst c-abl. When this compound (PD166326) was tested against variousp210^(Bcr-Abl) theramuteins, however, it showed activity against some ofthe mutants such as p210^(Bcr-Abl-E255K), but the p210^(Bcr-Abl-T315I)theramutein was found to remain 10 fold more resistant (Huron et al.2003, Table 3). Furthermore, in each case the compound was stillmarkedly less effective on the p210^(Bcr-Abl) theramuteins than it wasagainst the wild-type p210^(Bcr-Abl). When the compound was testedagainst p210^(Bcr-Abl-T315I) mutant activity, it was unable to inhibitthe activity to any appreciable extent (p. 1270, left hand column,second paragraph; see also FIG. 4.). Thus, the disclosed compound wasable to inhibit a theramutein that is partially resistant to STI-571,but had no activity against the T315I mutant of Bcr-Abl, which wasalready known at that time to be the theramutein that exhibited the mostresistance to STI-571. Hence purely and simply, the Huron methodologyfailed to identify an effective inhibitor of the p210^(Bcr-AblT315I)theramutein.

Indeed, prior to the disclosure of this invention, including both thedetailed methodology described for the first time herein as well as thecompositions provided herein, no one anywhere in the world has beensuccessful in identifying a chemical agent, let alone a methodology thatis capable of identifying a chemical agent that effectively inhibits thep210^(Bcr-AblT315I) theramutein to an equal or greater extent thanSTI-571 is able to do with respect to the wild type p210^(Bcr-Abl)protein. (See Shah et al., Science, July, 2004; O'Hare et al., Blood,2004; Tipping et al., Leukemia, 2004; Weisberg et al., Leukemia, 2004).

It cannot be overemphasized that such compounds would be immenselyuseful, because at the present time there is no alternative for patientswho progress to p210^(Bcr-Abl-T315I) theramutein-mediated imatinibmesylate-resistant status. Once patients develop such resistance, thereis no other effective alternative treatment available, and death iscertain. The method described herein provides the first reportedapproach to identify, pharmacologically characterize and chemicallysynthesize effective inhibitors of the p210^(Bcr-Abl-T315I) theramutein.Moreover, the skilled investigator will immediately recognize theapplicability and generalizability of this approach to any highlydrug-resistant theramutein. Finally, the skilled investigator willfurther recognize that linking a phenoresponse as defined herein to theincreased presence and functional activity of a particular POI in thecell under appropriate conditions allows one to utilize the method withany given endogenous target protein for which a therapeuticallyeffective compound is sought.

In the present invention, a test cell is used that displays a carefullyselected phenotypic characteristic (as defined below) which is linked tothe presence and functional activity of the particularprotein-of-interest (POI) or theramutein-of-interest (TOI) in the cellunder appropriate conditions. With respect to a theramutein, this shouldbe qualitatively the same as the phenotypic characteristic displayed bya cell that expresses the prototheramutein. A phenotypic characteristic(i.e. a non-genotypic characteristic of the cell) is a property which isobserved (measured), selected and/or defined for subsequent use in anassay method as described herein. Expression of the phenotypiccharacteristic is responsive to the total activity of the protein in thecell, and is a result of the absolute amount of the protein and itsspecific activity. Often, the phenotypic characteristic is observable asa result of elevated levels of protein activity and is not apparent incells that express low amounts of the protein, or if the protein is alsoa theramutein, then the phenotypic characteristic will often not beapparent in cells expressing low amounts of either the theramutein orits corresponding prototheramutein. Further, it can often bedemonstrated that the phenotypic characteristic is modulated bymodulating the specific activity of the protein with an inhibitor oractivator of the theramutein, although this is not always the case sincean inhibitor or activator of the TOI may not always be available at thetime the skilled investigator undertakes such a project. (However,clearly a known inhibitor or activator of a given prototheramutein willalways exist as a result of the intrinsic definition of the nature of atheramutein itself.) Thus, for the purpose of defining the phenotypiccharacteristic to be subsequently used with a given test cell for assaypurposes, the skilled investigator may also use a substance capable ofincreasing or decreasing the expression of the gene encoding a given POI(such as a theragene in the case of a theramutein), which will in turnlead to increases or decreases of the level of the correspondingtheramutein. This allows the skilled investigator to simulate theeffects of certain types of activators or inhibitors of the theramutein(such as a suicide inhibitor of the theramutein, which is a class ofchemical agent which binds irreversibly and covalently modifies the TOI,rendering it permanently inactive), without actually having access tosuch a compound, for the purposes of refining the appropriate phenotypiccharacteristic for subsequently establishing a useful cellular assaysystem. Examples known to one of ordinary skill that would be helpfulfor such purposes include the use of anti-sense DNA oligonucleotides,small interfering RNAs, other RNA interference-based methodologies, andvector constructs containing inducible promoter systems. In this manner,the selected phenotypic characteristic is linked to the activity of thetheramutein in the test cell. Notably for theramuteins, the selectedphenotypic characteristic is usually also displayed by a cell thatoverexpresses the prototheramutein and in which the phenotypiccharacteristic is modulated by known inhibitors or activators of theprototheramutein.

A phenotypic characteristic is simply a characteristic of a cell otherthan a genotypic characteristic of the cell. Except for the specificrequirements of a properly defined phenotypic characteristic asdisclosed herein for the purposes of creating useful cellular assaysystems according to the teachings of certain of the embodiments of theinvention, no other limitation of the term phenotypic characteristic ofany kind or nature is intended or appropriate in order to properly andeffectively practice the invention. Indeed, the skilled artisan must beable to select any characteristic of the cell that maximizes the utilityof establishing the proper cell-based assay for his or her needs. Thephenotypic characteristic can be quantitative or qualitative and beobservable or measurable directly (e.g., observable with the naked eyeor with a microscope), but most commonly the characteristic is measuredindirectly using standard automated laboratory equipment and assayprocedures which are known to those of skill in the art. The term“observable” means that a characteristic may be measured or is otherwisedetectable under appropriate conditions by any means whatsoever,including the use of any type of laboratory instrumentation available.The term “detectable” is not the same as “detected.” A characteristicmay be detectable to a skilled artisan without being detected at anygiven time, depending upon how the investigator chooses to design theassay system. For example, in searching for activators of a POI such asa prototheramutein (or theramutein), it may be desirable to have therelevant phenotypic characteristic detected only after the addition of aknown activator or test substance capable of activating the POI. Thisprovides the ability to maximize the intensity of the signal that isgenerated by the test cell in the assay.

Phenotypic characteristics include but are not limited to growthcharacteristics, transformation state, differentiation state, substratephosphorylation state, catalytic activity, ion flux across the cellmembrane (calcium, sodium, chloride, potassium, hydrogen ions, etc.), pHchanges, fluctuations of second messenger molecules or otherintracellular chemical species such as cAMP, phosphoinositides, cyclicnucleotides, modulations of gene expression, and the like. Thecharacteristic of the cell may be observable or measurable continuously(e.g., growth rate of a cell), or after a period of time (e.g., terminaldensity of a cell culture), or transiently (e.g., modulation of aprotein causes a transient change in phosphorylation of a substrate ofthe protein, or a transient flux in ion flow across the membrane, orelevations or reductions in intracellular cAMP levels). In certainembodiments, a selected phenotypic characteristic may be detected onlyin the presence of a modulator of the protein. No limitations areintended with respect to a characteristic that may be selected formeasurement. As used herein, the terms “characteristic of a cell” and“phenotypic characteristic”, and simply “characteristic”, when used torefer to the particular measurable property of the intact cell or asubcellular fraction of the cell following the treatment of a test cellwith a substance, are identical. For example, a phenotypiccharacteristic can be focus formation that becomes observable when acell that over expresses a selected protein is cultured in the presenceof an activator of the protein, or it may be a transient increase ordecrease in the level of an intracellular metabolite or ion, such ascAMP, calcium, sodium, chloride, potassium, lithium,phosphatidylinositol, cGMP, bicarbonate, etc. It is obvious to one ofordinary skill in the art that after a cell is exposed to a testsubstance, the characteristic so measured (assayed) may be determined ona sub-cellular fraction of the cell. However, the initial treatment ofthe cell with a substance, which thereby causes the substance to comeinto contact with the cell, must be performed on the intact cell, not asub-cellular fraction.

The characteristic selected for measurement within the cell must not bean intrinsic physical or chemical property of the protein (ortheramutein or prototheramutein) itself (such as the mere amount (mass)of the protein inside the cell), but rather must be a characteristicthat results from the activity of the protein (or theramutein orprototheramutein) inside the cell, thus affecting a characteristic ofthe cell which is distinct from the theramutein itself, as discussed indetail above. For example, where the theramutein is a protein kinasethat is capable of undergoing autophosphorylation, a process whereby theenzyme is capable of catalyzing the phosphorylation of itself bytransferring a terminal phosphate group from ATP onto itself, it wouldNOT be appropriate to select the phosphorylation state of the TOI as anappropriate phenotypic characteristic of the cell for measurement. Thisis because such a characteristic does not reflect the activity of theTOI on other cellular components. As the skilled investigator knows,autophosphorylation is not necessarily reflective of the activity of aprotein kinase in a cell, since mutants of protein kinases are knownthat retain enzymatic activity sufficient to undergoautophosphorylation, yet have lost the capability to engage in signaltransduction events within the cell. The classic paper by White et al.(1988) is both educational and noteworthy in this respect.

The term “responsive phenotypic characteristic” means a characteristicof the cell which is responsive to inhibitors or activators of a givenprotein (including, e.g., a prototheramutein or theramutein). The term“known therapeutic agent” is defined as any agent that has beenadministered to a human being for the treatment of a disease in acountry of the world.

A useful phenotypic characteristic, as exemplified herein in associationwith p210^(Bcr-Abl) and theramuteins thereof, is disregulation of cellgrowth and proliferation. It is noted that the same or similar assay maybe appropriate for use with many different proteins of interest. Forexample, disregulations of growth, proliferation, and/or differentiationare common phenotypic characteristics that may result fromoverexpression of a variety of different cellular proteins. It is animportant teaching of this invention that by overexpressing a selectedprotein in order to cause the appearance of such a phenotypiccharacteristic, the characteristic becomes linked to the presence,amount, and specific activity of that selected protein under suitableconditions, and this linkage allows the skilled investigator to identifyinhibitors or activators of a protein of interest (POI) as desired.Accordingly, the phenotypic characteristic is responsive to changes inthe level and/or specific activity of the selected protein. Such aresponsive phenotypic characteristic, when also demonstrated to beresponsive to a known modulator of the POI is referred to herein as a“phenoresponse.” In the special case of a theramutein which has no knownmodulator, a modulator of the prototheramutein must be utilized toestablish a phenorespone to be used with the theramutein. The conceptionand recognition of this highly useful property of a cell represents oneof the substantial advances of this invention over the prior art,including Applicant's own prior original work in the general area ofcell-based assays (U.S. Pat. Nos. 4,980,281; 5,266,464; 5,688,655;5,877,007). The identification and selection of the phenoresponseprovides the skilled investigator with a cellular assay system that isextremely sensitive in terms of its ability to identify inhibitors oractivators of the POI, and therefore identifies such chemical agentswith a much higher degree of assurance than any other related assaymethod disclosed in the prior art.

Though not always necessary, it will often be advantageous to employcells that express high levels of the POI, and to select a phenotypiccharacteristic that results from overexpression of the POI. This isbecause phenotypic characteristics linked to the functioning of the POIgenerally become more distinguishable (easier to measure) as a POI isoverexpressed to a greater extent. Further, phenoresponses that areobserved in response to modulators of the POI are often amplified as thefunctional level of the POI is increased. Expressed another way, theselected phenoresponse observed in cells that overexpress the protein(or theramutein) is particularly sensitive to modulators of the protein(or theramutein).

Preferably, the protein is stably expressed in a test cell. Stableexpression results in a level of the protein in the cell that remainsrelatively unchanged during the course of an assay. For example,stimulation or activation of a component of a signaling pathway may befollowed by a refractory period during which signaling is inhibited dueto down-regulation of the component. For proteins of the invention, suchdown-regulation is usually sufficiently overcome by artificiallyoverexpressing the protein. Expressed another way, the expression issufficiently maintained that changes in a phenotypic characteristic thatare observed during the course of an assay are due primarily toinhibition or activation of the protein, rather than a change in itslevel, even if down-modulation of the protein subsequently occurs. Forthese reasons, although stable expression of the protein is preferred,transfection followed by transient expression of the protein may beemployed provided that the selected phenotypic characteristic ismeasurable and the duration of the assay system is short relative to theprogressive decline in the levels of the transiently expressed proteinthat is to be expected in such systems over time. For these reasons,stably expressing cell lines are preferred (U.S. Pat. No. 4,980,281).

The term “cellular specificity” means the ability of a compound, at agiven concentration, to modulate a selected phenoresponse of the Testcell without affecting the Control Cell to the same extent, if at all.The term “cellular specificity gap” (“CSG”) means a measurement of theability of a selected compound to modulate the selected phenoresponsecorresponding to a given target protein (not limited to a theramutein)in a test cell relative to the ability of said compound to modulate thesame phenoresponse in a corresponding control cell. For the purposes ofapplying the CSG technique to non-theramutein endogenous targetproteins, the selected phenoresponse must have been previously definedusing a known inhibitor or activator of the target protein.

Determination of the CSG provides the skilled investigator with a methodof comparing the relative potential therapeutic value of differentcompounds within a group of compounds (two or more) by comparing theirrelative cross-reactivity with control cells irrespective of the potencyagainst the target protein of any given compound within the group.Compounds that exhibit the greatest “specificity” in their activityagainst test cells relative to control cells are generally the mostdesirable compounds, since a “wide” CSG will assist in selecting acompound that may reasonably be assumed to have minimal potential sideeffects in patients as compared to other compounds within theaforementioned group that have “narrow” CSGs. The effects of the CSGmeasurement are seen most easily when comparing cell-based assaygenerated dose-response curves in their entirety, however the followinghypothetical example is also instructive.

Consider the following table of hypothetical compounds and theircorresponding IC₅₀ values using a cell-free assay system. This exampleuses a protein kinase as the target protein. This is the sort ofsituation that investigators skilled in the art are faced with on adaily basis when dealing with the problem of trying to perform leadoptimization on a selected compound or group of compounds for thepurpose of identifying a potential optimized lead candidate compound forsubsequent pre-clinical (animal) and clinical studies.

TABLE 1 Cell-Free Purified Protein Kinase Inhibition Assay. IC₅₀ againstIC₅₀ against Target a Non-Target Compound Protein Kinase (nM) ProteinKinase (nM) IC₅₀ Ratio A 0.2 10,000 50,000 B 3 10,000 3,333 C 250 10,00040 D 500 10,000 20

A standard approach in the art at the present time is to identifycompounds that exhibit a high degree of potency with respect toinhibition of the target protein kinase's enzymatic activity in acell-free assay system without showing significant inhibitory activityagainst a distinct but closely related protein kinase. As the results ofthe cell-free assay system shown above in Table 1 indicate, compound Ais the most potent of the series of compounds (A, B, C, D) and alsoshows the largest difference between its IC₅₀ against the target proteinrelative to its effect on the non-target protein. For example, if onewere interested in identifying inhibitors of the Abl kinase, one mightuse another protein kinase, such as the EGF receptor, c-kit, or c-Src,as a “negative” control kinase in such an assay. As with c-Abl, all ofthese latter enzymes are tyrosine protein kinases. Indeed, it iscommonplace in the field at the present time to use so-called “panels”of protein kinases, including serine/threonine kinases, tyrosinekinases, and dual-specificity kinases, in order to identify compoundsthat inhibit as few protein kinases as possible (other than the targetprotein kinase itself). The reasoning behind this approach is that thefewer the number of kinases that are inhibited in a cell-free system bya given compound, the less likely the compound is to have untoward sideeffects in the patient. However, there is very little clinical evidencethat actually supports this view.

Furthermore, in some cases it has been argued by others that compoundsthat target more than one kinase may have additional therapeutic effectsas compared to those compounds that are highly specific for only asingle target protein. There is some evidence for this being true in thecase of imatinib, whose cross-reactivity with c-kit has resulted inbeneficial effects for patients with certain histologic types ofcarcinoma of the small intestine, as discussed previously herein.Despite this cross-reactivity with c-kit, however, imatinib displays ahigh degree of cellular specificity in the assay systems of the presentinvention, which is consistent with its high degree of clinical efficacyand relatively modest side effect profile within the first three yearsof treatment. However, as the specificity of a given compound drops inthe cellular systems of the present invention, the increasedcross-reactivity with other targets such as (in this example) otherprotein kinase family members may result in untoward side effects in thepatient. This is discussed in further detail below.

TABLE 2 Results from Applying the Method(s) of the Invention byUtlilizing a Phenoresponse-Based Cellular Assay System and Measuring theCellular Specificity Gap (CSG) IC₅₀ against Compound IC₅₀ against TestCells (nM) Control Cells (nM) CSG A 1 1 1 B 10 100 10 C 500 20,000 40 D10 200 20

The conclusion that would be drawn by the investigator from the resultsof the cell-free assay shown in Table 1 above is that compound A is themost potent compound, showing a 50% inhibitory concentration (IC₅₀)value of 0.2 nanomolar (0.2 nM). As shown in Table 2 and FIG. 14,however, this compound, shows no specificity for the Test cells relativeto the Control Cells, since it's IC₅₀ for the Control cells is also 1nanomolar. Similarly, compounds B and D are still quite potent, withboth having IC₅₀'s of 10 nM against the Test cells, whereas compound Dis more specific than B since its IC₅₀ for the Control cells is higher(200 nM). The CSG measurements of compounds B and D immediately reflectthat compound D would be the preferred compound between these two, allother considerations being equal. Most importantly, however, is thatcompound C is shown in this example to be the best compound of the groupin terms of its CSG, at 40 (Table 2, FIG. 15). This means that compoundC shows the greatest specificity for the Test cells relative the Controlcells, and would be expected to have the lowest incidence of inducingunwanted side effects in patients, since this finding is derived from adirect testing of the compound in a living cellular system, the ultimatepoint of pharmacological action for the overwhelming majority ofmedicines. The important point in this example is that the CSGmeasurement allows the skilled investigator to rank order the potentialtherapeutic value of a series of compounds independently of theirpotency in cell-free systems. Thus, although compound C is the leastpotent compound, it is the most specific in its ability to inhibit theTest cells while leaving the Control cells relatively unaffected overwide concentration range. This is reflected in its CSG of 40, anddemonstrates that compound C, rather than compound A, is the compoundthat should be given the highest priority for further pre-clinical andclinical development efforts.

Use of the method of present invention in this manner provides for anability to rank order and prioritize the pharmaceutical discovery anddevelopment process in a manner which was not possible previously.Through iterative application of the approach given above, the skilledinvestigator working together with medicinal chemists may synthesizeanalogus of compounds that score positively in the assay systemsdescribed herein, test such compounds in the assay methods of theinvention, rank order the compounds according to their CSG values,select the best ones for further development, and repeat the process asmany times as necessary in order to fully develop and optimize compoundsthat exhibit a high degree of specificity against a given Test cellrelative to its corresponding Control cell. Once a given compound hasbeen optimized using the system described herein, which generally meansthat the CSG between Control and Test cells (if measured using thecellular IC₅₀ ratio method described above) is at least three to fivefold, the skilled investigator can then proceed to complete the leadoptimization process through additional chemical modifications of thecompound selected in this way and tested for properties such as plasmahalf-life, oral bioavailability, and related parameters usingappropriate animal models. Of course, the likelihood of such compoundshaving the desired effects on the target protein in the cellularenvironment is virtually assured as a result of the process ofoptimizing said compounds according to the methods described herein,rather than with older, cell-free methods.

It will be immediately apparent to the skilled investigator thatanalogous approaches may also be utilized with activators of a giventarget protein. It will also be apparent to the skilled investigatorthat there are other ways of determining the CSG. For example, using theinhibitor example given above, another useful approach to determine theCSG is to measure the ratio of the highest concentration of a compoundwhich results in 50% growth inhibition of the control cell line, dividedby the lowest concentration of the compound at which at least 90% of thetest cell line is inhibited. Other obvious modifications of thisapproach may be utilized as well, including computing the logarithm ofthe concentrations at which compounds show a given percentage ofactivity, normalization of either the control or test cell responsesrelative to one another, etc. No limitation is intended on the nature ofthe computed or observed comparison of the control cell responsivenessto the test cell responsiveness for the purposes of determining the CSG.

If one uses the IC₅₀-ratio of control cells/test cells method asdescribed above, then compounds with CSG values less than or equal to 1would not generally be considered to be good clinical candidatecompounds, whereas compounds with CSG values of greater thanapproximately 10 would be quite promising and worthy of furtherconsideration.

This example also highlights the distinctions between the effects of agiven compound in a cell-free system versus the more medically andphysiologically relevant cell-based system of the present invention.

In an embodiment of the invention, compound profiling is used toidentify and/or minimize side effects associated with administration ofa compound to a patient. The cell-based lead optimization method allowsearly identification of potential side effects as compared to thecell-free approach. For example, imatinib shows a wide cellularspecificity gap according to the methods of the invention describedherein. This is consistent with imatinib's significant advance in thearea of anti-cancer drugs. However, it is not without side effects.Recent evidence demonstrates that imatinib is associated with cardiactoxicity in a small percentage of patients (Kerkeli et al., 2006). Thisgroup may increase over time as patients take imatinib for longerperiods of time.

Through the use of the methods of the invention, FIG. 16 shows thatimatinib tested at various concentrations on the wild type Ba/F3 cellline shows a slight but significant growth inhbitory effect atconcentrations that are substantially below the apparent IC₅₀ forcellular toxicity (about 1 μM) on the control cell line that imatinibexhibits at markedly higher concentrations. Such results become evenmore evident when the comparison of the effects of other compounds onthe Control cell line are compared to those of imatinib.

In still another implementation of the method, compounds which may showpromising activity in a cell-free system but have small CSG values (asdiscussed above) would be expected to have higher potential side effectsin patients, especially over longer treatment periods. Compounds havinglow CSG values have been reported by others with respect to certaintargets such at p210 Bcr-Abl^(T315I) mutant (Carter et al., 2005), andstill other groups have even entered such compounds into clinicaltrials. However, based upon the teachings of this invention it may beexpected that such compounds will have an increased incidence ofuntoward side effects in patients.

A preferred drug screening method of the present invention involves thefollowing:

1) Identification of a protein of interest (POI), such as a theramuteinfor which a novel inhibitor or activator is desired. Often, the POI isimplicated or suspected to be implicated in establishment or maintenanceof a disease state, perhaps due to inappropriate expression or amutation-induced change in specific activity. Identification of anappropriate theramutein, for example, may be performed using standardtechniques (See, Gorre et al., Science, 2001; see also PCT/US02/18729).Briefly, patients that have been given a course of a therapeuticallyeffective treatment using an activator or inhibitor of a known orsuspected prototheramutein and have subsequently shown clinical signsand symptoms consistent with disease relapse are identified, and cellsor tissue samples derived from such patients are obtained. Usingstandard laboratory techniques such as RT-PCR, the sequence of theprototheramutein is determined and compared to the previously determinednucleic acid sequence of the known prototheramutein gene or cDNAsequence. Mutations, if present, are identified and are correlated withfunctional resistance of the prototheramutein's function either incell-based or, more commonly, cell-free assay systems, again usingstandard methodology. Once resistance-inducing mutations are confirmed,then said one or more confirmed mutants comprise a defined theramuteinwhich may be used in the subsequent methods as described herein.

2) Provision of a test cell that expresses the POI and displays anobservable (measurable) phenotypic characteristic that is linked toexpression of the POI. In the case of a theramutein, the phenotypiccharacteristic is usually one which has been previously shown to beresponsive to inhibitors or activators of the theramutein or, morecommonly, the corresponding prototheramutein. Such a phenotypiccharacteristic that is linked to expression of the POI and has beenpreviously shown to be responsive to inhibitors or activators of the POI(or the prototheramutein-of-interest (pTOI)) is defined herein as a“phenoresponse.” One embodiment of this invention is the definitive useof the phenoresponse for the purpose of identifying compounds that arelikely to be inhibitors or activators of the TOI. This may beaccomplished through the use of a high-throughput screen using a cellline overproducing a given TOI and for which an appropriatephenoresponse has been identified and characterized. Alternatively, onemay utilize a high-throughput primary screen using a more genericphenotypic characteristic of a cell line (that does not qualify as aphenoresponse according to the teachings herein) and then utilize asecondary screen according to the teachings herein to distinguishbetween compounds that are true positive “hits”, i.e. inhibitors oractivators of the theramutein of interest, from false positive compoundsthat are not inhibitors or activators of the theramutein of interest. Inone embodiment, a cell is selected that naturally expresses thetheramutein such that a responsive phenotypic characteristic is presentunder suitable culture conditions which are obvious to one of ordinaryskill in the art. In other embodiments, the theramutein isoverexpressed, in some instances in a host cell that does not otherwiseexpress the theramutein at all. This usually involves construction of anexpression vector from which the theramutein can be introduced into asuitable host cell and overexpressed using standard vector systems andmethodology. (Gorre et al., 2001; Housey et al., 1988). In oneembodiment, overexpression results in a level of the theramutein that isat least about 3 times the amount of the protein usually present in acell. Alternatively, the amount is at least about 10 times the amountusually present in a cell. In another embodiment, the amount is at leastabout 20 times or more preferably at least about 50 times the amountusually present in a cell.

3) Provision of a control cell that expresses the POI to a lesser extentor not at all (e.g., an unmodified host cell or host cell harboring anexpression vector that does not express the POI). In the case of atheramutein of interest, the control cell can also be a cell expressingthe prototheramutein corresponding to the theramutein of interest.

As some of the muteins that are described herein are also enzymes, theyusually retain catalytic activity, and therefore the control cellusually displays substantially the same phenotypic characteristic as thetest cell. The phenotypic characteristic need not be quantitativelyalike in both cells, however. For example, a mutation that leads toreactivation of the prototheramutein may also increase, decrease, orotherwise affect its specific activity with respect to one or more ofits substrates in the cell. As a result, it may exhibit the selectedphenotypic characteristic to a greater or lesser extent. Accordingly, itmay be desirable in some cases to adjust expression of either or both ofthe prototheramutein and the theramutein such that test and controlcells exhibit the phenotypic characteristic to approximately the samedegree. This may be done, for example, by expressing the proteins frompromoters whose activity can be adjusted by adjusting the amount ofinducer present, all using standard methodology (see, for example,Sambrook et al. 1989 and 2001).

It will be obvious to one of ordinary skill in the art that a properlydefined phenoresponse may be quantitatively different between theprototheramutein- and the theramutein-expressing cell lines as a resultof differences in the specific activity (if any) between the theramuteinand its corresponding prototheramutein. Theramutein-inducing mutationsmay increase or decrease the specific activity of said theramuteinrelative to the corresponding prototheramutein. When comparing atheramutein expressing cell line with a prototheramutein expressing cellline, it is preferable that the selected phenoresponse is qualitativelythe same in both cell types. Thus, the skilled investigator may chooseto normalize the activity of the theramutein-expressing cell line tothat of the prototheramutein-expressing cell line, or vice versa. Suchnormalization methods are standard in the art. See, for example, Bolstadet al. (2003).

Alternatively, the skilled investigator may also wish to use unmodifiedhost cells or host cells harboring the expression vector only as controlcells for certain experimental procedures. (The host cells are the cellsinto which an expression vector encoding the theramutein was introducedin order to generate the test cells.) This may be the case where theinvestigator is only interested in identifying a specific inhibitor oractivator of the theramutein of interest, irrespective of whether or notsaid compound is also effective against the prototheramutein of interest(pTOI).

4) The test and control cells are then maintained or propagated(although not necessarily at the same time) in growth media (or even inintact animals) under suitable conditions such that the phenoresponsemay be expressed and assayed. Control cells that are expressing theprototheramutein may be treated with a known modulator of theprototheramutein, or with a test substance, and test cells are treatedwith test compounds to determine whether they are active against thetheramutein, as measured by the ability of said substances to modulatethe phenoresponse in the expected manner. Alternatively, control cellsnot expressing the prototheramutein may also be substituted, dependingupon the particular phenoresponse that the skilled investigator haschosen for study. Substances may then be assayed on the test cells and,optionally, on the control cells at the same time, or at another time,and the results compared.

In one embodiment of the invention, substances that are active withregard to the test cells can be rapidly identified by their ability tomodulate the phenoresponse of the test cells in the same manner as, forexample, the known modulator of the prototheramutein alters thephenoresponse of prototheramutein-expressing control cells. In anotherembodiment, active substances may be identified by their ability tomodulate the activity of the theramutein in the test cells while havinglittle or no effect on the unmodified (prototheramutein and/ortheramutein non-expressing) control cells. The skilled investigator willreadily appreciate the many variations of this approach that may beutilized to identify, for example, modulators that are more effectiveagainst the theramutein, or that are equally effective against both theprototheramutein and one or more corresponding specific theramuteins.

Other phenoresponses can be observed and/or measured and include, forexample, detection of substrates of the prototheramutein, and detectionof gene expression changes that are regulated by the activity of thetheramutein. In the simplest terms, any characteristic of the cell thatthe skilled investigator has previously correlated with the functionalactivity of the theramutein may be suitable for use with such methods.However, in selecting a given characteristic, the skilled investigatormust first verify that said characteristic fulfills the criteria ofbeing a phenoresponse according the teachings as given in detail herein.The skilled investigator may also wish to normalize the phenoresponsewith the theramutein expressing cells to that of the prototheramuteinexpressing cells.

Characteristics suitable for detection may be measured by a variety ofmethods very well known to those of skill in the art. Such methodsinclude, but are not limited to, detection of fluorescence of suitablylabeled proteins (FACS), immunohistochemistry (IHC) for detection ofprotein expression, competitive radioligand binding assays, solid matrixblotting techniques, such as Northern, Southern, and Western blots ofcell extracts, reverse transcriptase polymerase chain reaction (RT-PCR),enzyme linked immunosorbent assays (ELISA), phosphorylation assays, gelretardation assays, membrane potential perturbations, and the like. Therelevant phenotypic characteristic may be detected either on the intactcell after treatment with a test substance or, alternatively, on asubcellular fraction of the cell after treatment of the intact cell witha test substance.

Once compounds are identified that have the desired effect on thetheramutein expressing test cells, it may be desirable (but notnecessary) to independently verify that the compounds identified areexerting their effects on the theramutein through a direct bindingmechanism, i.e. that the compounds fulfill the criteria of beinginhibitors or activators (as desired) of the theramutein according tothe teachings of the invention (the reader is referred to thedefinitions of the terms “activator” and “inhibitor” as given above).This may be accomplished with numerous standard binding assays that areknown to one of ordinary skill in the art, involving either purifiedprotein samples or intact cellular binding assays using cellstransfected with the appropriate prototheramutein or theramuteintogether with appropriate controls as dictated by sound scientificmethods. Since such methods are well established in the art they willnot be reiterated here. Numerous reference texts comprehensively discusssuch techniques (see, for example, Foreman and Johansen, 2002; Enna S.J. et al. (1991) Current Protocols in Pharmacology, Wiley & Sons,Incorporated; Bonifacino, J. S. et al. (1999) Current Protocols in CellBiology, Wiley & Sons, Incorporated). See also Housey, G. M. 1988,Chapter 4, and references therein; see also Horowitz et al., 1981.

In a particular embodiment of the invention, the method is used toidentify substances that are inhibitors of the p210^(Bcr-Abl-T315I)theramutein. The prototheramutein and theramutein are each expressed inBa/F3 (murine) cells using standard methodology and the phenoresponsesthat are observed are growth characteristics (terminal cell density fora carefully defined cell culture, and growth in the absence ofInterleukin-3 (IL-3). Unmodified host cells, or host cells containingthe expression vector only or both, may optionally also be used. Instill another embodiment, the test cells alone may be used with orwithout reference to a known inhibitor or activator.

Another useful assay is the determination of the state ofphosphorylation of a direct substrate of p210^(Bcr-Abl-T315I). One suchsubstrate is Crkl (Gorre et al., Science 293:876-80 (2001)), an adapterprotein which mediates the connection between Bcr-Abl and Ras. Thephosphorylation state of CRKL is representative of the signalingactivity of p210^(Bcr-Abl) in a cell. Another downstream substrate isp62DOK. Any such substrate would suffice for these purposes, provided ofcourse that phosphorylation of said substrate has been shown to occurinside the cell, and is not simply an autophosphorylation event of theTOI or PTOI as discussed above. Other signal transduction cascadecomponents may also be monitored, including src family kinases, STATS,PI3 Kinase, rafkinase, RAS, MEK, ERK1 and ERK2, JNK1, 2 and 3, MLK1, 2and 3, MKK4, MKK7, AKT, mTOR, HSP90, and others.

As exemplified herein, inhibitors of the T315I theramutein have beenidentified. Furthermore, these inhibitors are also active to differingextents against the wild type prototheramutein p210^(Bcr-Abl-wt).

According to the present invention, a therapeutically effective amountof one or more compounds that modulate the functional activity of ap210^(Bcr-Abl) theramutein is administered to a mammal in need thereof.The term “administering” as used herein means delivering the compoundsof the present invention to a mammal by any method that may achieve theresult sought. They may be administered, for example, orally,parenterally (intravenously or intramuscularly), topically,transdermally or by inhalation. The term “mammal” as used herein isintended to include, but is not limited to, humans, laboratory animals,domestic pets and farm animals. “Therapeutically effective amount” meansan amount of a compound that, when administered to a mammal, iseffective in producing the desired therapeutic effect, such asinhibiting kinase activity, inhibiting cancer cell growth and division,etc.

The invention provides a method of treating disease in a mammal byadministering to the mammal an effective amount of a modulator of atheramutein. Suitable diseases to be treated according to the presentinvention include, but are not limited to, relapsing neoplastic or otherproliferative disorders that have become resistant to previouslyadministered drugs. The method is also useful for overcoming variationamong individuals with respect to susceptibility to drug treatment thatresults from allelic differences among therapy targets. For example, therole of p210^(Bcr-Abl) tyrosine kinase signaling in CML has beenextensively demonstrated, as has the role of theramuteins ofp210^(Bcr-Abl) in drug resistant recurrence of CML. Further, differentmuteins of p210^(Bcr-Abl) exhibit varying sensitivity to inhibitors ofp210^(Bcr-Abl). Although some theramuteins arise during drug therapy,others may preexist in the population. These latter examples will not berecognized as theramuteins until such time as the disease state ensuesand is followed by treatment with a known class of therapeutic agents.Only after said treatment will such preexisting theramuteins revealthemselves as being clinically significant in terms of relativenon-responsiveness leading to the progression of the disease in thepatient harboring the theramutein.

In an embodiment of the invention, theramutein modulators areadministered in combination with one or more other anti-neoplasticagents. Any suitable anti-neoplastic agent can be used, such as achemotherapeutic agent, radiation or combinations thereof. Theanti-neoplastic agent can be an alkylating agent or an anti-metabolite.Examples of alkylating agents include, but are not limited to,cisplatin, cyclophosphamide, melphalan, and dacarbazine. Examples ofanti-metabolites include, but not limited to, doxorubicin, daunorubicin,and paclitaxel, gemcitabine, and topoisomerase inhibitors irinotecan(CPT-11), aminocamptothecin, camptothecin, DX-8951f, topotecan(topoisomerase I inhibitor), and etoposide (VP-16; topoisomerase IIinhibitor) and teniposide (VM-26; topoisomerase II inhibitor). When theanti-neoplastic agent is radiation, the source of the radiation can beeither external (external beam radiation therapy—EBRT) or internal(brachytherapy—BT) to the patient being treated. The dose ofanti-neoplastic agent administered depends on numerous factors,including, for example, the type of agent, the type and severity of thetumor being treated and the route of administration of the agent. Itshould be emphasized, however, that the present invention is not limitedto any particular dose, route of administration, or combination ofchemotherapeutic agents or other therapeutic regimens that are combinedwith the administration of protein modulators.

Anti-neoplastic agents which are presently known in the art or beingevaluated can be grouped into a variety of classes including, forexample, mitotic inhibitors, alkylating agents, anti-metabolites,intercalating antibiotics, growth factor inhibitors, cell cycleinhibitors, enzymes, topoisomerase inhibitors, anti survival agents,biological response modifiers, anti-hormones, and anti-angiogenesisagents, all of which can be administered with inhibitors or activatorsof theramuteins.

A modulator of a theramutein can be administered with antibodies thatneutralize other receptors involved in tumor growth. Further, amodulator of a theramutein can be administered with a compound thatotherwise modulates a component of a signal transduction pathway,preferably a component of the signal transduction pathway in which thetheramutein is active and which is common to one or more other signaltransduction pathways. In an embodiment of the invention, a theramuteinmodulator is used in combination with a receptor antagonist that bindsspecifically to the Epidermal Growth Factor Receptor (EGFR).Particularly preferred are antigen-binding proteins that bind to theextracellular domain of EGFR and block binding of one or more of itsligands and/or neutralize ligand-induced activation of EGFR. An EGFRantagonist can be an antibody that binds to EGFR or a ligand of EGFR andinhibits binding of EGFR to its ligand. Ligands for EGFR include, forexample, EGF, TGF-α, amphiregulin, heparin-binding EGF (HB-EGF) andbetacellulin. EGF and TGF-α are thought to be the main endogenousligands that result in EGFR-mediated stimulation, although TGF-α hasbeen shown to be more potent in promoting angiogenesis. It should beappreciated that the EGFR antagonist can bind externally to theextracellular portion of EGFR, which can or can not inhibit binding ofthe ligand, or internally to the tyrosine kinase domain in the case ofchemical agents. Examples of EGFR antagonists that bind EGFR include,without limitation, biological agents such as antibodies (and functionalequivalents thereof) specific for EGFR, and chemical agents (smallmolecules), such as synthetic kinase inhibitors that act directly on thecytoplasmic domain of EGFR.

Other examples of growth factor receptors involved in tumorigenesis arethe receptors for vascular endothelial growth factor (VEGFR-1 andVEGFR-2), platelet-derived growth factor (PDGFR), nerve growth factor(NGFR), fibroblast growth factor (FGFR), and others.

In a combination therapy, the theramutein inhibitor is administeredbefore, during, or after commencing therapy with another agent, as wellas any combination thereof, i.e., before and during, before and after,during and after, or before, during and after commencing theanti-neoplastic agent therapy. For example, the theramutein inhibitorcan be administered between 1 and 30 days, preferably 3 and 20 days,more preferably between 5 and 12 days before commencing radiationtherapy. In a preferred embodiment of the invention, chemotherapy isadministered prior to, concurrently with or, more preferably, subsequentto antibody therapy.

In the present invention, any suitable method or route can be used toadminister theramutein inhibitors of the invention, and optionally, toco-administer anti-neoplastic agents and/or antagonists of otherreceptors. The anti-neoplastic agent regimens utilized according to theinvention, include any regimen believed to be optimally suitable for thetreatment of the patient's neoplastic condition. Different malignanciescan require use of specific anti-tumor antibodies and specificanti-neoplastic agents, which will be determined on a patient to patientbasis. Routes of administration include, for example, oral, intravenous,intraperitoneal, subcutaneous, or intramuscular administration. The doseof antagonist administered depends on numerous factors, including, forexample, the type of antagonists, the type and severity of the tumorbeing treated and the route of administration of the antagonists. Itshould be emphasized, however, that the present invention is not limitedto any particular method or route of administration.

Suitable carriers include, for example, one or more of water, saline,phosphate buffered saline, dextrose, glycerol, ethanol and the like, aswell as combinations thereof. Carriers can further comprise minoramounts of auxiliary substances, such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the theramutein modulator as the active ingredient. The compositionscan, as is well known in the art, be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the mammal.

The compositions of this invention can be in a variety of forms. Theseinclude, for example, solid, semi-solid and liquid dosage forms, such astablets, pills, powders, liquid solutions, dispersions or suspensions,liposomes, suppositories, injectable and infusible solutions. Thepreferred form depends on the intended mode of administration andtherapeutic application.

Such compositions of the present invention are prepared in a manner wellknown in the pharmaceutical art. In making the composition the activeingredient will usually be mixed with a carrier, or diluted by a carrierand/or enclosed within a carrier which can, for example, be in the formof a capsule, sachet, paper or other container. When the carrier servesas a diluent, it can be a solid, semi-solid, or liquid material, whichacts as a vehicle, excipient or medium for the active ingredient. Thus,the composition can be in the form of tablets, lozenges, sachets,cachets, elixirs, suspensions, aerosols (as a solid or in a liquidmedium), ointments containing, for example, up to 10% by weight of theactive compound, soft and hard gelatin capsules, suppositories,injection solutions, suspensions, sterile packaged powders and as atopical patch.

It should be appreciated that the methods and compositions of thepresent invention can be administered to any suitable mammal, such as arabbit, rat, or mouse. More preferably, the mammal is a human.

The compounds according to the invention may also be present as salts.In the context of the invention, preference is given to pharmaceuticallyacceptable salts. Pharmaceutically acceptable salts refers to an acidaddition salt or a basic addition salt of a compound of the invention inwhich the resulting counter ion is understood in the art to be generallyacceptable for pharmaceutical uses. Pharmaceutically acceptable saltscan be salts of the compounds according to the invention with inorganicor organic acids. Preference is given to salts with inorganic acids,such as, for example, hydrochloric acid, hydrobromic acid, phosphoricacid or sulfuric acid, or to salts with organic carboxylic or sulfonicacids, such as, for example, acetic acid, maleic acid, fumaric acid,malic acid, citric acid, tartaric acid, lactic acid, benzoic acid, ormethanesulfonic acid, ethanesulfonic acid, phenylsulfonic acid,toluenesulfonic acid or naphthalenedisulfonic acid. Pharmaceuticallyacceptable salts can also be metal or ammonium salts of the compoundsaccording to the invention. Particular preference is given to, forexample, sodium, potassium, magnesium or calcium salts, and also toammonium salts which are derived from ammonia or organic amines, suchas, for example, ethylamine, di- or triethylamine, di- ortriethanolamine, dicyclohexylamine, dimethylaminoethanol, arginine,lysine, ethylenediamine or 2-phenylethylamine. (see, Berge et al. J.Pharm. Sci. 1977, 66, 1-19).

Throughout this application, various publications, reference texts,textbooks, technical manuals, patents, and patent applications have beenreferred to. The teachings and disclosures of these publications,patents, patent applications and other documents in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which the present invention pertains.

It is to be understood and expected that variations in the principles ofinvention herein disclosed may be made by one skilled in the art and itis intended that such modifications are to be included within the scopeof the present invention.

The following examples further illustrate the invention, but should notbe construed to limit the scope of the invention in any way. Detaileddescriptions of conventional methods, such as those employed in theconstruction of vectors and plasmids, the insertion of genes encodingpolypeptides into such vectors and plasmids, the introduction ofplasmids into host cells, and the expression and determination thereofof genes and gene products can be obtained from numerous publications,including Sambrook, J et al., (1989) Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Cold Spring Harbor Laboratory Press; Coligan, J. etal. (1994) Current Protocols in Immunology, Wiley & Sons, Incorporated;Enna, S. J. et al. (1991) Current Protocols in Pharmacology, Wiley &Sons, Bonifacino, J. S. et al. (1999) Current Protocols in Cell Biology,Wiley & Sons, and U.S. Pat. No. 4,980,281. All references mentionedherein are incorporated in their entirety.

EXAMPLES

It is to be understood and expected that variations in the principles ofthe invention herein disclosed may be made by one skilled in the art andit is intended that such modifications are to be included within thescope of the present invention.

Examples of the invention which follow are set forth to furtherillustrate the invention and should not be construed to limit theinvention in any way.

Example 1 Identification of a Protein Modulator

p210^(Bcr-Abl-T315I) is a theramutein of the p210Bcr-Abl protein(p210^(Bcr-Abl)) that is resistant to inhibition by imatinib mesylate(Gleevec, STI-571). The mutation at position 315 converts a threonine toan isoleucine residue and is one of several mutations that are observedamong resistant or relapsed patients. This particular mutant, however,is the most resistant such theramutein yet identified.

A phenoresponse was determined for a Ba/F3 cell line engineered tooverexpress the p210^(Bcr-Abl-T315I) theramutein. The phenoresponse wasdetermined relative to non-transformed Ba/F3 cells and Ba/F3 cells thatexpress the p210^(Bcr-Abl-wt) prototheramutein. The phenoresponse wasthe ability of the T315I mutants to grow to a higher cell saturationdensity under analogous culture conditions as compared to the controlnon-transformed Ba/F3 cell line, and to grow in the absence ofinterleukin 3 (IL-3), which is required for maintenance of the controlnon-transformed Ba/F3 cell line. The phenoresponse was defined andcharacterized according to the teachings given above.

The detection system utilized was a high speed cell imaging and countingsystem in which 3 μl sample volumes of cells were sequentially injectedthrough a 5 μl optical microcell, digitally imaged and electronicallystored, scanned, and then counted, all under a microcomputer-basedcontrol system. The system has the capacity to perform direct cellcounts on samples from cultures as small as 500 μl and providesstatistically significant total cell counts from culture samplescontaining as few as 12,500 cells. All of the figures displaying cellcount and viability assays utilized this system for data acquisition andanalysis. Simultaneously with the cell count performed, the system isalso capable of determining overall cell viability by distinguishingcounted, imaged cells that have excluded trypan blue (counted as“viable” cells) from cells which have taken up the trypan blue dye(counted as “non-viable” cells). Injection of trypan blue into the cellsample occurs immediately prior to the sample being sequentiallyinjected into the microcell for simultaneous cell counting and imaging.

The system may be integrated into the workflow of high-throughputscreening devices to provide a sensitive and precise cell counting andcell viability assay system that is more reliable and less prone toconfounding effects of metabolic viability-based cellular assays such asXTT or Alamar blue.

Initially, approximately 113,000 compounds were screened atconcentrations generally ranging from 10 to 2 μM to identify a subsetthat was capable of affecting growth of Ba/F3 cells (Ba/F3 T315I cells)overexpressing the p210^(Bcr-Abl-T315I) theramutein by any means.

A total of approximately 11,760 compounds showed greater than 50% growthinhibition, which were thought to correspond to approximately 4500distinct chemical classes. Retesting of these compounds with the samecell line yielded a database of compound responsiveness which was thensorted and rank ordered according to those compounds exhibiting thehighest overall growth inhibition. From this rank ordered database, thehighest scoring 130 compounds (based upon the greatest degree of growthinhibition observed at the lowest concentrations that compounds weretested) were then rescreened in a defined cell-based assay system usingBa/F3 T315I as test cells and wild type Ba/F3 as control cells accordingto the methods of the present invention. Compounds of interest werethose that differentially inhibited growth of Ba/F3 cells expressing thep210^(Bcr-Abl-T315I) theramutein relative to non-transformed wild typeBa/F3 cells. Six compounds were identified that fulfilled the desiredcriteria, and some of these compounds were analyzed in further detailusing the Ba/F3 p210^(Bcr-Abl-wt) cells line (Ba/F3 P210 cells) as well.One compound was unavailable for further testing due to lack ofavailability of additional material from the chemical supplier. Theremaining five compounds were independently evaluated in additionalcell-based assays using the aforementioned cell lines as well as in acell-free purified protein kinase assay using human recombinantlyproduced 120 Kd kinase domain fragments isolated from both wild typeP210 Bcr-Abl as well as P210 T315I mutant kinase domain.

All five compounds inhibited p210^(Bcr-Abl-T315I) 120 Kd activity asmeasured by inhibition of autophosphorylation activity. Thus, of the 6highest scoring compounds out of more than 113,000 compounds screened,at least 5 of the six directly inhibited the p210^(Bcr-AblT315I) mutantdirectly. One compound appeared to spread the recombinant protein bandout on the SDS page gel. This was also evident on the silver-stained gel(data not shown). It is possible that this compound may actually be a“suicide” inhibitor that is able to covalently cross-link the POI inorder to permanently inhibit its activity, but this will require furtherstudy.

Taken together, the teachings and the results described herein provideconclusive proof that the system is capable of identifying inhibitors oractivators of the selected theramutein, and the skilled investigatorwill immediately recognize that such a system may be easily applied toany other theramutein or other protein with only obvious, minormodifications.

Representative examples of the cell-based assay results demonstratingselective inhibition of growth of the Ba/F3 T315I cell line relative tothe wild type non-transformed Ba/F3 cells are shown in FIGS. 1 and 2.The compounds inhibited growth and reduced the viability of cellsexpressing the T315I theramutein at concentrations under which thegrowth and viability of the wild type Ba/F3 non-transformed cells (notexpressing either p210^(Bcr-Abl-wt) or p210^(Bcr-Abl-T315I)) wererelatively unaffected, whereas cells expressing both theprototheramutein as well as the theramutein were substantiallyinhibited. In some instances, the T315I expressing cells were inhibitedto an even greater extent than the P210 prototheramutein expressingcells. (See, for example, FIG. 3, right hand side, Compound 3 resultsagainst P210 and T315I cells.

In summary, the methods presented herein provide a fundamental advancein the form of a generalizable approach for creating or identifyingmodulators of any given theramutein. The results demonstrateconclusively the power of the method to identify critically neededcompounds to overcome a specific type of acquired drug resistance thatis uniformly fatal in certain patient populations and is presentlyuntreatable. Furthermore, it is evident to one of skill in this art thatthe techniques and methods described herein may, using obviousmodifications, be straightforwardly generalized to any potentialtheramutein or other disease associated protein of clinicalsignificance.

It is remarkable that out of a primary screen of more than 100,000compounds where approximately 10,000 compounds exhibited some degree ofgrowth inhibition, when the most potent growth inhibitory substanceswere rescreened using the Method described in detail herein, 6 distinctcompounds were identified and all of the compounds that weresubsequently tested exhibited inhibitory activity in a cell-freepurified protein kinase assay using the T315I mutant (one compound wasunavailable for further testing). Based upon such remarkable results, itbecomes immediately clear to the skilled artisan that the method may beeffectively applied toward the identification of inhibitors oractivators of any protein based upon the proper selection and definitionof the phenoresponse according to the teachings in the sections givenabove and the documents incorporated by reference herein. For example,with knowledge of the foregoing, one of ordinary skill in the art couldeasily design an assay system to identify inhibitors of theramuteinsderived from other prototheramuteins known to exhibit mutations thatconfer drug resistance such as the c-kit gene product or the EpidermalGrowth Factor (EGF) Receptor (EGFR), or the Platelet Derived GrowthFactor (PDGF) Receptor α and β. No limitation should be inferred uponthe utility of the method with respect to its ability to be utilizedwith any given protein, including theramuteins and protothermuteins,expressed in any mammalian cell type for which a correspondingphenoresponse is detectable.

Example 2 Phenoresponse-Based Optimization of a Protein Modulator

In this example, a compound previously identified according to theteachings of this invention is optimized for activity against itsselected protein target. However, unlike methods typically used in theprior art, the optimization process herein is also performed entirelythrough the use of the phenoresponse-based cellular assay system. Forcompleteness sake and to demonstrate the power of the methodology torefine, a cell-free assay system using recombinantly produced targetenzyme is also used to independently demonstrate that the compounds thatscore positively in the phenoresponse-based cellular assay indeed alsoscore positively in a cell-free assay system format that is standard inthe art and uses recombinantly produced enzyme.

Compound C2 which was originally identified as an inhibitor of the T315Itheramutein was subjected to a novel lead optimization program asfollows. Various chemical modifications were introduced into the basicscaffold structure of compound C2 using standard medicinal chemistrysynthetic methods. Once synthesized, the various analogues (chemicalvariants) were tested using the phenoresponse-based cellular assaysystem described above in Example 1.

Based upon the original structure of compound C2, the contribution(s) topharmacological activity arising from the phenyl ring that contained thebromo, chloro, and hydroxyl substituents were analyzed. An initialseries of analogues were synthesized that consisted of either theunsubstituted phenyl ring (C2-01), or various substituents on the phenylring, such as bromo, chloro, and hydroxyl, etc. located variouspositions around the phenyl ring. Detailed chemical structures are shownin Table 3.

TABLE 3 Optimization of C2 Compounds

C2

C2-27

C2-21

C2-01

C2-02

C2-05

C2-86

C2-87

C2-109

C2-112

C2-121

C2-122

C2-128

These compounds were then tested in the phenoresponse-based cellularassay system, as shown in FIG. 17. Each of the compounds was also testedin a standard cell free protein kinase autophosphorylation assay at aconcentration of 2 μM as shown in FIG. 18. As a comparison of FIG. 17and FIG. 18 will indicate, there was a striking, essentially completequalitative correlation between the activity of the compounds in thephenoresponse-based (cellular) assay system and their correspondingactivity in the cell free purified protein kinase autophosphorylationassay.

Additional chemical modifications were made to the same phenyl ringdiscussed above for a variety of medicinal chemistry purposes and forreasons that are beyond the scope of this invention, but were related toenhancing potency against the p210^(Bcr-Abl-T315I) target whilesimultaneously limiting cross-reactivity with the control wild typeBa/F3 non-transformed cells (not expressing either p210^(Bcr-Abl-wt) orp210^(Bcr-Abl-T315I)), as well as to improve selectivity, minimizepotential side effects in the patient, etc. As shown in FIGS. 17 and 18taken together with Table 3, additional compounds synthesized and testedincluded C2-109, C2-112, C2-122, and C2-128. A detailed comparison ofFIGS. 17 and 18 reveals once again that all of the compounds that scorepositively in the cellular assay system also exhibited protein kinaseinhibitory activity in the cell free system, whereas those that wereessentially inactive in the phenoresponse-based cellular assay were alsoessentially inactive in the cell-free system. These results conclusivelydemonstrate that the compounds showing inhibitory activity in thephenoresponse-based assay system were entirely consistent qualitativelywith the results obtained in the cell free protein kinaseautophosphorylation assay.

Furthermore, where there are modest differences in relative potencybetween the cell free assay results and the phenoresponse-based assayresults (see for example, compound C2-122 which appears to be morepotent in th cell-free assay than in the phenoresponse-based cellularsystem, such distinctions point out the enhanced ability of the cellularassay system to predict in vivo efficacy of a given compound as comparedto classical cell-free assay systems. Using a classical cell-freescreen, one might have considered C2-122 to be an important compound,yet the phenoresponse-based assay immediately rules it out as being lesspotent than several of the other compounds tested. This type of leadoptimization strategy employing a cellular system without dependence andreliance upon a cell-free radioligand or other binding assay has notbeen reported before in the prior art.

In summary, the present invention provides the skilled investigator witha powerful and rapid method for lead optimization which supplants thenecessity for repeated cell free in vitro verifications of the abilityof a compound to hit its corresponding target. Thus the optimizationprocess may be performed essentially entirely with reliance upon thephenoresponse-based assay systems results, obviating the need forrepetitive confirmatory cell free assay determinations. While suchconfirmatory experiments may be performed if the skilled investigatorchooses to do so, they are generally unnecessary with this method, asthe results given above unequivocally demonstrate.

The skilled investigator is well aware that no assay system of any kindor nature, whether it be a radioligand binding assay, ELISA, ligandbinding assay, or cellular assay, is free of false-positive results.This assay system, while surprisingly robust, will not be free of thepossibility of false positive results either, and the skilledinvestigator knows that independent verification of unusual results issimply good science and should be taken into consideration whereappropriate.

Example 3 Phenoresponse-Based Profiling of a Protein Modulator

The phenoresponse-based assay system of the invention can be used toprofile the biological activity of a given compound with respect to itsability to inhibit or activate multiple distinct protein targets todiffering extents. For example, in certain instances the skilled artisanmay be interested in identifying or optimizing modulators of a giventarget protein where additional proteins are known that are distinct buthighly related to the target POI. Such protein families may consist oftwo or more members that share a high degree of homology at both the DNAand amino acid sequence levels, yet the family members may have distinctfunctions within the cell. Through iterative application of thephenoresponse-based system described herein, one could create individualTest Cells expressing each of the distinct family members and thenutilize three or four or more distinct Test Cell lines withcorresponding defined phenoresponses to identify or optimize compoundsthat are selective for one particular family member.

In yet another embodiment of the present invention, the skilled artisanmay also choose to express two or three or even four distinct proteintargets in a single Test Cell (or Test Cell line) and create aphenoresponse-based assay system useful for identifying compounds thatare NOT selective among individual isozymes of a given protein family.In certain therapeutic situations, lack of selectivity among individualfamily members may be preferable. Ibuprofen, for example, is anestablished, low-cost safe and effective non-steroidal anti-inflammatorydrug that does not significantly discriminate between the cyclooxygenasetype 1 (COX-1) and COX-2 family members. Such lack of discrimination mayin some instances be beneficial and may reduce the likelihood of certainunwanted side effects that may occur with an overly selective chemicalagent.

Profiling the biological effects of a given compound with respect to itsability to inhibit or activate certain related protein targets, whetheror not such targets are members of the same protein family, also hassubstantial value from the perspective of understanding the molecularand cellular mechanism(s) of action of a given chemical agent. Forexample, in the case of imatinib, not only does the compound inhibit thewild type version of the P210 Bcr/Abl protein (p210^(Bcr-Abl-wt)), italso cross reacts with and is capable of inhibiting the c-kitoncoprotein as well. As discussed above in the background of theinvention, this cross-reactive inhibition of the kit oncoprotein isserendipitous, because gastrointestinal stromal tumors (GIST), a type oftumor arising in the small intestine, are driven by kit activity and arethus responsive to imatinib treatment as well (NEJM paper). Thus, suchcross reactivity with other related proteins need not always beassociated with toxicity of a drug. In some instances suchcross-reactivities can be therapeutically effective.

Representative Compounds of the Invention corresponding to the variouschemical formulae given above were tested in the cellular assay systemdescribed elsewhere herein (see Example 1) and assigned activitycategories as shown in Table 4. The assigned activity categories arerepresented by the following designations, wherein the IC₅₀ for a givencell line is the concentration at which a given compound inhibits thegrowth of that cell line by 50% in the cellular assay system. Compoundstested on a given cell line that exhibited an IC₅₀ value that was <300nM (less than 300 nanomolar) were designated as Category “A” compounds.Compounds tested on a given cell line that exhibited an IC₅₀ value thatwas <1 μM (less than imicromolar) were designated as Category “B”compounds. Compounds tested on a given cell line that exhibited an IC₅₀value that was <10 μM (less than 10 micromolar) were designated asCategory “C” compounds. Compounds tested on a given cell line thatexhibited an IC₅₀ value that was >1 μM (greater than or equal to 10micromolar) were designated as Category “D” compounds.

TABLE 4 Structure: wt BaF3 T315I

B A

C C

B B

B B

D D

D D

D D

B A

B B

D D

C B

B B

D D

A A

D D

A A

A A

B B

B A

A A

B B

D D

B A

B B

C B

B B

D D

C B

A A

A A

A A

A A

A A

B B

C C

B B

Reaction Scheme:

Experimental Details:

The mixture of compound 1 (25 g) and N, N-dimethylaniline (24.2 g) inPOCl₃ (110 mL) was refluxed for 5 h. POCl₃ was removed by evaporation atreduced pressure and the residue was poured into ice-water (500 g)cautiously and stirred for 1 h. The mixture was then filtered and thesolid was washed with water to give compound 2 as a yellow solid.

2. Reaction Scheme:

Experimental Details:

To a solution of compound 2 (1.04 g) in 15 ml of ethanol, 1.08 g (2 eq)of morphine was added dropwise at −10° C. in 15 min. The mixture wasstirred for 0.5 h and heated at 50° C. for 15 min. After cooling anddilution with water (50 ml), compound 3 was obtained as a yellow solidpowder after filtration.

3. Reaction Scheme:

Experimental Details:

To 1.1 g of compound 3 was added 8 ml of NH₂NH₂.H₂O. The mixture wasrefluxed for 2 h. After cooling, the crude product was obtained afterfiltration. Purification by column chromatography gave pure compound 4as a light yellow solid.

4. Reaction Scheme:

Experimental Details:

To the solution of compound 5 (1.0 g, 1.0 eq) and DMF (0.05 g, cat.amount) in 20 mL of dichloromethane was added (COCl)₂ (0.81 g, 1.1 eq)dropwise. The reaction mixture was stirred at r.t. for 2 h and thenconcentrated to give 1.2 g of crude product of compound 6, which wasused for the next step without further purification.

5. Reaction Scheme:

Experimental Details:

To the crude product of compound 6 (1.2 g, 1.0 eq) in 20 mL ofdichloromethane was added 3-trifluoromethyl-phenylamine (0.94 g, 1.0 eq)and triethylamine (0.71 g, 1.2 eq). The reaction mixture was stirred atr.t. overnight, washed with 1 N NaOH solution, 1 N HCl solution andbrine. The organic layer was collected, dried over Na₂SO₄, andconcentrated to give crude product of compound 7. After purification bycolumn chromatography, 1.1 g of compound 7 was obtained.

6. Reaction Scheme:

Experimental Details: To the mixture of compound 7 (0.3 g, 1.0 eq) and 3mL of trifluoroacetic acid was added hexamethylenetetramine (0.53 g, 4.0eq). The reaction mixture was immediately sealed and heated to 90° C.for 20 h. After cooling, the reaction mixture was adjusted to pH 8 with1 N NaOH solution, extracted with dichloromethane and the organic phasewas dried, concentrated to give a brown solid. Purification bypreparative TLC gave compound 8 as a yellow solid.

7. Reaction Scheme:

Experimental Details:

The mixture of compound 4 (30 mg, 1.0 eq) and compound 8 (19 mg, 1.0 eq)in 5 mL of dichloromethane was stirred at r.t. overnight. Theprecipitates were collected and washed with dichloromethane thoroughly,dried under vacuum to give the desired compound.

Example 5

1. Reaction Scheme:

Experimental Details:

To the mixture of compound 1 (1.0 g, 1.0 eq), compound 2 (0.92 g, 1.0eq), Na₂CO₃ (0.77 g, 1.5 eq) in 15 mL of dioxane was added Pd(PPh₃)₄(0.56 g, 0.1 eq), and the reaction mixture was refluxed under N₂ for 16h. After cooling, the mixture was filtered and the filtrate wasevaporated to dryness and purified by column chromatography to givecompound 3.

2. Reaction Scheme:

Experimental Details:

To the mixture of compound 3 (0.3 g, 1.0 eq) and 2 mL of trifluoroaceticacid was added hexamethylenetetramine (0.62 g, 4.0 eq). The reactionmixture was immediately sealed and heated to 90° C. for 20 h. Aftercooling, the reaction mixture was adjusted to pH 8 with 1 N NaOHsolution, extracted with dichloromethane and the organic phase wasdried, concentrated to give a brown solid. Purification by preparativeTLC gave compound 4 as a yellow solid.

3. Reaction Scheme:

Experimental Details:

The mixture of compound 4 (30 mg, 1.0 eq) and compound 5 (19 mg, 1.0 eq)in 5 mL of dichloromethane was stirred at r.t. overnight. Theprecipitates were collected and washed with dichloromethane, dried undervacuum to give the desired compound.

Example 6

1. Reaction Scheme:

Experimental Details:

To the solution of compound 1 (0.6 g, 1.0 eq) in 10 mL of methanol wasadded 4 mL of 1 N NaOH solution, and the mixture was stirred at r.t.overnight. Solvent was evaporated and the residue was acidified to pH 6with 5% citric acid, extracted with dichloromethane. The organic layerwas dried, concentrated to give compound 2.

2. Reaction Scheme:

Experimental Details:

The mixture of compound 2 (0.4 g, 1.0 eq), trifluoromethyl phenylamine(0.39 g, 1.0 eq), EDC (0.71 g, 1.5 eq), HOBt (33 mg, 0.1 eq) in 10 mL ofdichloromethane was stirred at r.t. overnight. The mixture was washedwith 1 N NaOH solution, water, extracted with dichloromethane. Theorganic layer was dried over Na₂SO₄, concentrated to dryness, andpurified by column chromatography to give compound 3.

3. Reaction Scheme:

Experimental Details:

The solution of compound 3 (0.2 g, 1.0 eq) in 10 mL of dioxane wastreated with 4 mL of 1 N HCl, and the mixture was heated to 60° C. for 2h. After cooled, pH was adjusted to 8 by addition of NaHCO₃. The mixturewas extracted with dichloromethane, washed the organic layer with water,dried with Na₂SO₄ and evaporated to dryness. The crude product waspurified by column chromatography to give compound 4.

4. Reaction Scheme:

Experimental Details:

The mixture of compound 4 (40 mg, 1.0 eq) and compound 5 (30 mg, 1.0 eq)in 5 mL of dichloromethane was stirred at r.t. overnight. The mixturewas concentrated to dryness and purified by preparative HPLC to give thedesired compound.

Example 7

1. Reaction Scheme:

Experimental Details:

The mixture of compound 2 (0.3 g, 1.0 eq), 2-chloro-6-methyl-phenylamine(0.26 g, 1.0 eq), EDC (0.53 g, 1.5 eq), HOBt (25 mg, 0.1 eq) in 10 mL ofdichloromethane was stirred at r.t. overnight. The mixture was washedwith 1 N NaOH solution, water, and extracted with dichloromethane. Theorganic layer was dried over Na₂SO₄, concentrated to dryness, purifiedby column chromatography to give compound 3.

2. Reaction Scheme:

Experimental Details:

The solution of compound 3 (0.2 g, 1.0 eq) in 10 mL of dioxane wastreated with 4 mL of 1 N HCl, and the mixture was heated to 60° C. for 2h. After cooling, the pH was adjusted to 8 by addition of NaHCO₃. Themixture was extracted with dichloromethane, washed the organic layerwith water, dried with Na₂SO₄ and evaporated to dryness. The crudeproduct was purified by column chromography to give compound 4.

3. Reaction Scheme:

Experimental Details: The mixture of compound 4 (40 mg, 1.0 eq) andcompound 5 (30 mg, 1.0 eq) in 5 mL of dichloromethane was stirred atr.t. overnight. The mixture was concentrated to dryness and purified bypreparative HPLC to give the desired compound.

Example 8

1. Reaction Scheme:

Experimental Details:

To a solution of 1 g (5.5 mmol) of 5-bromo-2-cyanopyridine, 0.97 g of(6.05 mmol 1.1 eq) 3-(trifluoromethyl)aniline in 100 ml of toluene wasadded 3 eq of t-BuONa, 0.2 eq of BINAP and 0.1 eq of Pd₂(dba)₃. Then thesolution was heated to reflux overnight. The reaction was monitored byLC/MS. The volatiles were removed under reduced pressure. The crudeproduct was purified by flash chromatography to afford compound 2.

2. Reaction Scheme:

Experimental Details:

250 mg (0.95 mmol) of compound 2 was added to 20 mL of concentrated HCl,then the solution was heated to reflux until the starting materialdisappeared. The mixture was concentrated under reduced pressure toobtain compound 3 as a yellow solid without purification.

3. Reaction Scheme:

Experimental Details:

A solution of 50 mg (0.18 mmol) of compound 3 in 3 mL of dichloromethanewas added to 0.5 mL of thionyl dichloride. The mixture was heated andstirred for 3 h. Finally the solution was evaporated under reducedpressure. Compound 4 was obtained and used in next step withoutpurification.

4. Reaction Scheme:

Experimental Details:

A solution of 50 mg of compound 4 and 43 mg (1.2 eq) of hydrazine in 5mL of DCM was stirred at 25° C. for 3 hours. The reaction mixture wasconcentrated and the residue was purified by preparative HPLC to givethe desired compound.

Example 9

1. Reaction Scheme:

Experimental Details:

A suspension of compound 1 (25 g, 0.145 mol) and SeO₂ (27.5 g, 0.247mol) in acetic acid (1200 mL) was heated to reflux for 12 h. Thereaction mixture was concentrated under reduced pressure to dryness. Theresidue was dissolved into water and brought to pH=9 by adding K₂CO₃.The resulted mixture was extracted with EA (100 mL×3). The combined EAwas dried over Na₂SO₄. After filtrating off the Na₂SO₄, the filtrate wasconcentrated under reduced pressure to give the crude product 2, whichwas used in next step without purification.

2. Reaction Scheme:

Experimental Details:

A solution of 2 above prepared in ethanol triethyl orthoformate (10 mL)was refluxed 4 h. After removing off the solvent, the residue wasseparated by column to give the product 3 as oil.

3. Reaction Scheme:

Experimental Details:

To a stirred and degassed mixture of compound 3 (73 mg, 0.28 mmol) andcompound 4 (50 mg, 0.28 mmol), and _(t)BuONa (27 mg, 0.56 mmol) andBINAP (70.4 mg, 1.12 mol) in toluene (15 mL) was added Pd₂(dba)₃ (26 mg,0.028 mmol) under N₂ atmosphere and stirred at 80° C. for 48 h. Afterfiltering off the solid, the filtrate was concentrated to give the crudeproduct 5 which was used in the next step without purification.

4. Reaction Scheme:

Experimental Details:

A solution of compound 5 (335 mg, 0.1 mmol) in dichloromethane (10 mL)was treated with BBr₃ (146 mg, 0.6 mmol) at −30° C. under N₂ atmosphere,then was stirred at room temperature for 4 h. The reaction was pouredunto ice-water and then was brought by adding Na₂CO₃. The resultingmixture was extracted with dichloromethane (25 mL×3), the combinedorganic layer was dried over Na₂SO₄. After filtrating off the Na₂SO₄,the filtrate was concentrated to give the crude product 6 which was donenext step without purification.

5. Reaction Scheme:

Experimental Details:

A solution of compound 6 (27.74 mg, 0.1 mmol) and compound 7 (21 mg, 0.1mmol) in anhydrous CH₂C₂ (300 mL) was stirred under reflux for 6 h. Thesolvent was removed under reduced pressure. The residue was separated byprep-TLC to give the desired compound.

Example 10

1. Reaction Scheme

Experimental Details:

To a stirred and degassed mixture of compound 1 (5 g, 25 mmol) andcompound 2 (3.4 g, 27 mmol) in DMF (50 mL) and aqueous Na₂CO₃ (20 mL, 2M) was added Pd₂(dbbf)₃ (26 mg, 0.028 mmol) under N₂ atmosphere andstirred at 100° C. for 18 h. After cooling to room temperature andfiltrating off the solid, the filtrate was extracted with EA (200 mL).The organic layer was concentrated to dryness. The residue was purifiedby column to give the crude product 3.

2. Reaction Scheme:

Experimental Details:

A mixture of compound 3 (2 g, 0.1 mol) and Na₂S₂O₄ (5.2 g, 0.3 mol) inmethanol (80 mL) and H₂O (20 mL) was heated to reflux for 3 h. Thereaction was concentrated to dryness under reduced pressure. The residuewas dissolved into water and then was extracted with EA (150 mL). Theorganic layer was washed with brine twice and dried over Na₂SO₄. Afterfiltrating off the Na₂SO₄, the filtrate was concentrated to give theproduct 4.

3. Reaction Scheme:

Experimental Details:

To a stirred and degassed mixture of compound 5 (228 mg, 88 mmol) andcompound 4 (150 mg, 88 mmol), and _(t)BuONa (170 mg, 176 mmol) and BINAP(210 mg, 176 mmol) in toluene (25 mL) was added Pd₂(dba)₃ (79 mg, 0.88mmol) under N₂ atmosphere and stirred at 80° C. for 48 h. Afterfiltering off the solid, the filtrate was concentrated to give the crudeproduct 6.

4. Reaction Scheme:

Experimental Details:

A solution of compound 6 (140 mg, 4 mmol) in dichloromethane (20 mL) wastreated with BBr₃ (600 mg, 10.6 mmol) at −30° C. under N₂ atmosphere,then was stirred at room temperature for 4 h. The reaction was pouredunto ice-water and then was brought pH=9 by adding Na₂CO₃. The resultingmixture was extracted with dichloromethane (25 mL×3), the combinedorganic layer was dried over Na₂SO₄. After filtrating off the Na₂SO₄,the filtrate was concentrated to dryness. The residue was purified bycolumn to give the product 7.

5. Reaction Scheme:

Experimental Details:

A solution of compound 7 (98 mg, 0.36 mmol) and compound 8 (64 mg, 0.3mmol) in anhydrous CH₂Cl₂ (300 mL) was stirred under reflux for 6 h. Thesolvent was removed under reduced pressure. The residue was separated byprep-TLC to give the desired compound.

Example 11

1. Reaction Scheme:

Experimental Details:

To a stirred and degassed mixture of compound 1 (5.9 g, 25 mmol) andcompound 2 (3.3 g, 27 mmol) in DMF (50 mL) and aqueous Na₂CO₃ (20 mL, 2M) was added Pd₂(dbbf)₃ (26 mg, 0.028 mmol) under N₂ atmosphere andstirred at 100° C. for 18 h. After cooling to room temperature andfiltrating off the solid, the filtrate was extracted with EA (200 mL).The organic layer was concentrated to give the crude product 3 which isdone next step without purification.

2. Reaction Scheme:

Experimental Details:

A mixture of 3 (6.5 g, 27.9 mmol) and Pd(OH)₂ (10%, 0.5 g) in ethanol(200 mL) was stirred under hydrogen atmosphere (20 psi) at roomtemperature for 2 hour. The catalyst was filtrated off, and the filtratewas removed under vacuum to afford the product 4 as a colorless oil.

3. Reaction Scheme:

Experimental Details:

To a stirred and degassed mixture of compound 4 (406 mg, 20 mmol) andcompound 5 (520 mg, 20 mmol), and _(t)BuONa (170 mg, 176 mmol) and BINAP(210 mg, 176 mmol) in toluene (25 mL) was added Pd₂(dba)₃ (79 mg, 0.88mmol) under N₂ atmosphere and stirred at 80° C. for 48 h. Afterfiltrating off the solid, the filtrate was concentrated to give thecrude product 6 which is used in next step without purification.

4. Reaction Scheme:

Experimental Details:

A solution of compound 6 (383 mg, 10 mmol) in dichloromethane (20 mL)was treated with BBr₃ (600 mg, 10.6 mmol) at −30° C. under N₂atmosphere, then was stirred at room temperature for 4 h. The reactionwas poured unto ice-water and then was brought to pH 9 by adding Na₂CO₃.The resulting mixture was extracted with dichloromethane (25 mL×3), thecombined organic layer was dried over Na₂SO₄. After filtrating off theNa₂SO₄, the filtrate was concentrated to dryness. The residue waspurified by column to give the product 7.

5. Reaction Scheme:

Experimental Details:

A mixture of 7 (60 mg 022 mmol) nicotinaldehyde (33 mg, 0.15 m mol) indichloromethane (10 mL) was heated to reflux for 3 hr. After removingoff solvent, the residue was purified by chromatography to give thedesired compound.

Example 12

1. Reaction Scheme:

Experimental Details:

A solution of DMAP (9.3 g, 0.077 mol) and compound 1 (10 g, 0.051 mol)and Boc₂O (12 g, 0.051 mol) in tBuOH (200 mL) was stirred at roomtemperature overnight. The solvent was removed under reduced pressureand the residue was purified by flash chromatography on silica gel.(ethyl acetate/petroleum ether=10:1) to give 2 as a colorless oil.

2. Reaction Scheme:

Experimental Details:

A mixture of 2 (6.17 g, 27.9 mmol) and Pd(OH)₂ (10%, 1 g) in ethanol(200 mL) was stirred under hydrogen atmosphere (50 psi) at roomtemperature for 4 hour. The catalyst was filtrated off, and the filtratewas removed under vacuum to afford the product 3 as a colorless oil.

3. Reaction Scheme:

Experimental Details:

A mixture of compound 3 (2.65 g, 12 mmol) and compound 4 (2.6 g, 10mmol) and _(t)BuONa (1.34 g, 14 mmol,) and Pd₂(dba)₃ (46.5 mg, 50 mmol,)and DCHPB (70 mg, 0.2 mmol) in dry toluene (50 mL) was heated to 80-90°C. under N₂ for 24 hours. The precipitation was filtrated and thefiltrate was removed in vacuo and the residue was purified bychromatography on silica gel (ethyl acetate/petroleum ether=10:1) togive afford 5 as a yellow oil.

4. Reaction Scheme:

Experimental Details:

To a solution of 5 (0.9 g, 2.24 mmol) in CHCl₃ (50 mL), CF₃COOH (40 mL)was added at 0° C. After addition complete, the resulting mixture wasstirred at room temperature overnight. The solvent was removed underreduced pressure to dryness. The residue was recrystallized from etherto yield an off-white solid. The solid was dissolve in ammonia (10 mL).The mixture was brought the pH=7.0 by adding 1M HCl and precipitated.The precipitate was collected and washed with cold water (5 mL), anddried under reduced pressure to afford 6 as a dark solid.

5. Reaction Scheme:

Experimental Details:

A mixture of 6 (60 mg, 0.22 mmol), nicotinaldehyde (33 mg, 0.15 m mol)in dichloromethane (10 mL) was heated to reflux for 3 hr. After removingoff solvent, the residue was purified by chromatography to give thedesired compound as a yellow solid.

Example 13

1. Reaction Scheme:

Experimental Details:

To a stirred and degassed mixture of compound 1 (6.7 g, 25 mmol) andcompound 2 (4.1 g, 27 mmol) in DMF (50 mL) and aqueous Na₂CO₃ (20 mL, 2M) was added Pd₂(dbbf)₃ (26 mg, 0.028 mmol) under N₂ atmosphere andstirred at 100° C. for 18 h. After cooling to room temperature andfiltrating off the solid, the filtrate was extracted with EA (200 mL).The organic layer was concentrated to give the crude product 3.

2. Reaction Scheme:

Experimental Details:

A mixture of 3 (3.2 g, 10 mmol) and Pd(OH)₂ (10%, 0.5 g) in ethanol (200mL) was stirred under hydrogen atmosphere (20 psi) at room temperaturefor 2 hour. The catalyst was filtrated off, and the filtrate was removedunder vacuum to afford the product 4 as colorless oil.

3. Reaction Scheme:

Experimental Details:

To a stirred and degassed mixture of compound 4 (297 mg, 10 mmol) andcompound 5 (259 mg, 10 mmol), and _(t)BuONa (170 mg, 17.6 mmol) andBINAP (210 mg, 17.6 mmol) in toluene (25 mL) was added Pd₂(dba)₃ (79 mg,0.88 mmol) under N₂ atmosphere and stirred at 80° C. for 48 h. Afterfiltrating off the solid, the filtrate was concentrated to give thecrude product 6 which is done next step without purification.

4. Reaction Scheme:

Experimental Details:

A solution of compound 6 (446 mg, 10 mmol) in dichloromethane (20 mL)was treated with BBr₃ (600 mg, 10.6 mmol) at −30° C. under N₂atmosphere, then was stirred at room temperature for 4 h. The reactionwas poured unto ice-water and then was brought pH=9 by adding Na₂CO₃.The resulting mixture was extracted with dichloromethane (25 mL×3), thecombined organic layer was dried over Na₂SO₄. After filtrating off theNa₂SO₄, the filtrate was concentrated to dryness. The residue waspurified by column to give the product 7.

5. Reaction Scheme:

Experimental Details:

A mixture of 7 (67 mg, 0.15 mmol), nicotinaldehyde (33 mg, 0.15 m mol)in dichloromethane (10 mL) was heated to reflux for 3 hr. After removingoff solvent, the residue was purified by chromatography to give thedesired compound as a yellow solid.

Example 14

1. Reaction Scheme:

Experimental Details:

To a stirred and degassed mixture of compound 1 (3 g, 0.02 mol) andcompound 2 (3.4 g, 0.02 mol), and KOH (5.28 g, 0.1 mol) and TBBA (6.44g, 0.02 mol) in anhydrous THF (100 mL) was added Pd (PPh₃)₄ (2.31 g, 2mmol) under N₂ atmosphere and stirred under reflux for 12 h. Afterfiltrating off the solid, the filtrate was concentrated to dryness. Theresidue was purified by column to give the product 3, which is used innext step without purification.

2. Reaction Scheme:

Experimental Details:

To a stirred and degassed mixture of compound 3 (334 mg, 1.7 mmol) andcompound 4 (434 mg, 1.7 mmol), and t-BuONa (322 mg, 3.4 mmol) and BINAP(420 mg, 0.67 mol) in toluene (60 mL) was added Pd₂(dba)₃ (156 mg, 0.017mmol) under N₂ atmosphere and stirred at 80° C. for 48 h. Afterfiltrating off the solid, the filtrate was concentrated to dryness. Theresidue was purified by column to give the product 5.

3. Reaction Scheme:

Experimental Details:

A solution of compound 5 (100 mg, 0.3 mmol) in dichloromethane (10 mL)was treated with BBr₃ (393 mg, 0.6 mmol) at −30° C. under N₂ atmosphere,then was stirred at room temperature for 4 h. The reaction was pouredunto ice-water and then was brought by adding Na₂CO₃. The resultingmixture was extracted with dichloromethane (25 mL×3), the combinedorganic layer was dried over Na₂SO₄. After filtrating off the Na₂SO₄,the filtrate was concentrated to give the crude product 6.

4. Reaction Scheme:

Experimental Details:

A mixture of 6 (39 mg, 0.13 mmol), nicotinaldehyde (29 mg, 0.13 m mol)in dichloromethane (10 mL) was heated to reflux for 3 hr. After removingoff solvent, the residue was purified by chromatography to give thedesired compound as a yellow solid.

Example 15

1. Reaction Scheme

Experimental Details:

Paraformaldehyde (1.8 g, 59.3 mmol) was added to a solution of compound1 (1.0 g, 5.9 mmol) in acetic acid (40 mL), followed by NaCNBH₃ (1.8 g,28.8 mmol) at 10° C. After stirring of 16 h at room temperature, thesolution was poured into ice/water (100 mL), and the PH adjusted to 10with concentrated NaOH. The solution was extracted with DCM (3×100 mL).The combined organic layers were dried (MgSO₄), filtrated andconcentrated in vacuo to give compound 2.

2. Reaction Scheme:

Experimental Details:

0.84 g (4.3 mmol) of compound 2 was hydrogenated under 1 atm hydrogenwith Pd/C for 16 hour. The reaction mixture was filtered and thefiltrate was concentrated to afford compound 3 without furtherpurification.

3. Reaction Scheme:

Experimental Details:

A solution of 250 mg (1.5 immol) of compound 3, 0.2 eq of BINAP, 0.1 eqof Pd₂(dba)₃, 3 eq of Cs₂CO₃ and 5-Bromo-2-diethoxymethyl-pyridine(0.783 g, 3.01 mmol) in 10 mL of 1,4-dioxane was reacted at 150° C.under microwave for 2 hours. The reaction was monitored by LC-Ms. Themixture was concentrated and the residue was purified by preparative TLCto afford compound 4.

4. Reaction Scheme:

Experimental Details:

A solution of 300 mg (0.55 mmol) of compound 4 in 5 mL of DCM was added4 mL of TFA. The reaction mixture was stirred for 30 min at r.t. Themixture was added ice/water and basified by NaHCO₃ to PH=10 andextracted with DCM (15 mL*3). The combined organic layer was washed withwater and brine, dried over MgSO₄, filtered and concentrated to affordcompound 5.

5. Reaction Scheme:

Experimental Details:

A solution of 80 mg (0.295 mmol) of compound 5 and(5-Fluoro-4-morpholin-4-yl-pyrimidin-2-yl)-hydrazine (125 mg, 0.59 mmol)in 10 mL of DCM was stirred at 25° C. for 15 hours. The reaction mixturewas concentrated and the residue was purified by preparative HPLC toafford compound 6.

6. Reaction Scheme:

Experimental Details:

To a solution of compound 6 (40 mg, 0.086 mmol) in dry DCM (5 mL) wasadded drop wise BBr (22 mg, 0.258 mmol) at 0° C. The reaction mixturewas stirred for 3 h at r.t. The reaction was quenched with methanol, andthe mixture was concentrated. The residue was purified by preparativeHPLC to give the desired compound.

Example 16

Experimental Details:

To a stirred solution of 3-Bromoaniline (0.86 g) in 30 ml of toluenewere added 0.1 eq of Pd(PPh₃)₄, 5 ml of sat. aq. of Na₂CO₃ and asolution of was vigorously stirred under reflux for 15 h and cooled, 10ml of H₂O was added, and the Na₂SO₄ and concentrated. The residue waspurified by column chromatography

1. Reaction scheme:

Experimental details: To a stirred solution of 3-Bromoaniline (0.86 g)in 30 ml of toluene were added 0.1 eq of Pd(PPh₃)₄, 5 ml of sat. aq. ofNa₂CO₃ and a solution of 3-methoxyphenyl boronic acid (0.75 g) in 10 mlof EtOH under N₂ atmosphere. The mixture was vigorously stirred underreflux for 15 h and cooled, 10 ml of H₂O was added, and the mixture wasextracted with CH₂Cl₂ (20 ml×3). The combined organic layers were driedover Na₂SO₄ and concentrated. The residue was purified by columnchromatography (PE/EA=10:1) to get pure compound 2.

2. Reaction scheme:

Reaction Details:

A mixture of compound 2 (59.5 mg), 5-bromo-2-diethoxymethyl-pyridine(116.7 mg), t-BuONa (86.4 mg), BINAP (36.7 mg) and Pd₂(dba)₃ (27.4 mg)in dioxane (2 ml) was microwaved for 2 hs at 150° C., the solution wasfiltered and concentrated. The residue was purified by preparative TLCto afford compound 3.

3. Reaction scheme:

Reaction Details:

To a solution of compound 3 (120 mg) in 5 ml of DCM was added 1 ml ofBBr₃, the reaction was stirred overnight at r.t. Then added 5 ml of H₂Oto the mixture, extracted with EtOAc and concentrated. The crude productwas purified by pre. TLC to afford compound 4.

4. Reaction Scheme:

Experimental Details:

A mixture of 43.5 mg of compound 4 and 32 mg of hydrazine in 5 ml DCMwas stirred overnight at r.t. and concentrated. The crude product waspurified by prereparative TLC to afford the desired compound.

Example 17

1. Reaction Scheme:

Experimental Details:

A solution of 2.0 g (16.2 mmol, 1.0 eq) of compound 1, 0.05 eq of BINAP,0.05 eq of Pd₂(dba)₃, 1.2 eq of t-BuONa and 1-Bromo-3-nitro-benzene(3.28 g, 16.2 mmol, 1.0 eq) in 20 mL of anhydrous toluene was reacted at100° C. for 24 hours. The reaction was monitored by LC-MS. The mixturewas concentrated and the residue was purified by column chromatographyto afford compound 2.

2. Reaction Scheme:

Experimental Details:

2.5 g of compound 2 was hydrogenated under 1 atm of hydrogen with Pd/C(0.25 g) for 16 hour. The reaction mixture was filtered and the filtratewas concentrated to afford compound 3 without further purification.

3. Reaction Scheme:

Experimental Details:

A solution of 500 mg (2.33 mmol) of compound3,5-Bromo-2-diethoxymethylpyridine (607 mg, 2.33 mmol), 0.05 eq ofxantphos, 0.05 eq of Pd₂(dba)₃ and 1.5 eq of t-BuONa in 10 mL of toluenewas refluxed at 100° C. for 24 hours. The reaction was monitored byLC-MS. The mixture was concentrated and the residue was purified bycolumn

chromatography to afford compound 4.

4. Reaction Scheme:

Experimental Details:

100 mg of compound 4 was treated with 1 mL of HCl (1N aqueous solution)and 10 mL of dioxane. The mixture was stirred at rt for 4 h, adjusted topH 8-9 with 0.5 N NaOH solution. After extracted with DCM, dried organiclayer with Na₂SO₄ and concentrated to give compound 5.

5. Reaction Scheme:

Experimental Details:

A solution of 50 mg (0.16 mmol) of compound 5 and(5-Fluoro-4-morpholin-4-ylpyrimidin-2-yl)-hydrazine (50 mg, 0.23 mmol)in 5 mL of DCM was stirred at 25° C. for 15 hours. The reaction mixturewas concentrated and the residue was purified by preparative HPLC toafford compound 6.

6. Reaction Scheme:

Experimental Details:

To a solution of compound 6 (50 mg, 0.09 mmol) in dry DCM (5 mL) wasadded dropwise BBr₃ (20 mg, 0.25 mmol) at 0° C. The reaction mixture wasstirred for 3 hr at r.t. The reaction was quenched with methanol, andthe mixture was concentrated. The residue was purified by preparativeHPLC to give the desired compound.

Example 18

1. Reaction Scheme:

Experimental Details: A solution of 10 g (70.92 mmol) of3-fluoro-nitrobenzene, 1 eq imidazole and 2 eq K₂CO₃ in 100 ml of DMSOwas heated at 130° C. for 5 hours. The reaction was monitored by LC-MS.Then 500 mL of water was added and the precipitate was filtered and thesolid was washed with water and dried to afford compound 2 withoutfurther purification.

2. Reaction Scheme:

Experimental Details:

5 g (31.4 mmol) of compound 2 was hydrogenated under 1 atm hydrogen withPd/C for 0.5 hour. The reaction mixture was filtered and the filtratewas concentrated to afford compound 3 without further purification.

3. Reaction Scheme:

Experimental Details:

A solution of 500 mg (3.14 mmol) of compound 3, 0.1 eq of xantphose, 0.1eq of Pd₂(dba)₃ and 1.5 eq of t-BuONa in 10 mL of toluene was refluxedat 130° C. for 15 hours. The reaction was monitored by LC-Ms and washedwith water and extracted with EtOAc. The combined organic layer waswashed with brine and dried over MgSO₄. Filtered and concentrated,residue was purified by preparative TLC to afford compound 4.

4. Reaction Scheme:

Experimental Details:

A solution of 200 mg of compound 4 in 5 mL of 1,4-dioxane was added 8 mLof 4N HCl and heated at 80° C. for 2 hours. The reaction mixture wasbasified by 2N NaOH to ph=10 and extracted with DCM (15 mL*3). Thecombined organic layer was washed with water and brine, dried overMgSO₄, filtered and concentrated to afford 250 mg of crude product. Thecrude product was purified by preparative TLC to afford compound 5.

5. Reaction Scheme:

Experimental Details:

A solution of 80 mg of compound 5 and 1 eq of compound 6 in 5 mL of DCMwas stirred at 25° C. for 15 hours. The reaction mixture wasconcentrated and the residue was purified by preparative HPLC to affordthe desired compound.

Example 19

Experimental Details:

A solution of 1.50 g of 1-Bromo-3-methyl-5-nitro-benzene and 1.60 g (1.2eq) of Piperazine-1-carboxylic acid tert-butyl ester, 0.1 eq ofxantphos, 0.1 eq of Pd₂(dba)₃ and 1.5 eq of t-BuONa in 20 mL of toluenewas refluxed at 130° C. for 4 hours. The reaction was monitored by LC-Msand washed with water and extracted with EtOAc. The combined organiclayer was washed with brine and dried over Na₂SO₄. Filtered andconcentrated, residue was purified with column chromatography on silicagel using 10:1 PA:EA as an eluant. The appropriate fractions werecombined and concentrated under reduced pressure, to give intermediate1.

2. Reaction Scheme:

Experimental Details:

1.6 g of intermediate of 1 was hydrogenated under 1 atm hydrogen withRaney/Ni for 2 hours. The reaction mixture was filtered and the filtratewas concentrated to afford intermediate 2 without further purification.

3. Reaction Scheme:

Experimental Details:

A solution of 1.20 g of Intermediate of 2, and 1.30 g (1.20 eq) of5-Bromo-2-diethoxymethyl-pyridine, 0.1 eq of xantphose, 0.1 eq ofPd₂(dba)₃ and 1.5 eq of t-BuONa in 20 mL of toluene was refluxed at 130°C. for 4 hours. The reaction was monitored by LC-MS and washed withwater and extracted with EtOAc. The combined organic layer was washedwith brine and dried over Na₂SO₄. Filtered and concentrated, residue waspurified with column chromatography on silica gel using 4:1 PA:EA as aneluant. The appropriate fractions were combined and concentrated underreduced pressure to give intermediate 3.

4. Reaction Scheme:

Experimental Details:

200 mg of intermediate 3 was dissolved in 10 ml of DCM. 130 mg of TFAwas added to the reaction mixture solution dropwise and stirred for 3 hat r.t. The reaction mixture was based with NaHCO₃ solution to neutraland brine, dried over Na₂SO₄, filtered and concentrated to afford 220 mgof crude product. The crude product was purified by preparative TLC toafford intermediate 4.

5. Reaction Scheme:

Experimental Details:

A solution of 50 mg of intermediate 4 and 1.0 eq of(5-Fluoro-4-morpholin-4-ylpyrimidin-2-yl)-hydrazine in 5 mL of DCM wasstirred at r.t. for 3 hours. The reaction mixture was washed with waterand brine, concentrated it to give the residue and purified bypreparative HPLC to afford the desired compound.

1. Reaction Scheme:

Experimental Details:

15 ml of IM BH₃/THF was added dropwise into a solution of 3 g (13.95mmol) of 4-bromo-2-methyl-benzoic acid in 20 ml of THF at 0° C. Thereaction solution was allowed to reach room temperature for 1 hour andquenched by the dropwise addition of 50 ml of 50% aqueous THF. Themixture was treated with Na₂CO₃ and concentrated. The residue wasextracted with Et₂O. The organic layer was dried to give compound 2.

2. Reaction Scheme:

Experimental Details:

A solution of 2.4 g (11.9 mmol) of compound 2 in 20 ml of DCM was addeda slurry of 5.1 g (23.8 mmol) of PCC in 60 ml of DCM. The reactionsolution was stirred for 1 hour at r.t. diluted with 300 ml of Et₂O andfiltered. The filtrate was concentrated to give compound 3.

3. Reaction Scheme:

Experimental Details:

A solution of 2.1 g (14 mmol) was added into a solution of ethanol whichcontaining 1.9 g (9.55 mmol) of compound 3. The reaction solution washeated to reflux for 3 hours, and then concentrated. The solid waswashed with NaHCO₃ and extracted with acetic ether. The organic layerwas dried to give compound 4.

4. Reaction Scheme:

Experimental Details:

0.7 g of compound 4, 0.41 g of 3-trifluromethyl-phenylamine 0.32 g ofPd₂(dba)₃, 0.21 g of binap and 0.02 g of t-BuONa were added into 35 mlof toluene. The reaction solution was heated to reflux overnight, andconcentrated. The crude product was purified by column chromatography(ethyl acetate/hexane=1:1) to give compound 5.

5. Reaction Scheme:

Experimental Details:

The solution of compound 5 (0.2 g, 1.0 eq) in 10 mL of dioxane wastreated with 4 mL of 1 N HCl, and the mixture was heated to 60° C. for 2h. After cooling, the pH was adjusted to 8 by addition of NaHCO₃. Themixture was extracted with dichloromethane, washed the organic layerwith water, dried with Na₂SO₄ and evaporated to dryness. The crudeproduct was purified by column chromatography to give compound 6.

6. Reaction Scheme:

Experimental Details:

A solution of 80 mg of compound 6 and 1 eq of compound 7 in 5 mL of DCMwas stirred at 25° C. for 15 hours. The reaction mixture wasconcentrated and the residue was purified by preparative HPLC to affordthe desired compound.

Example 21

1. Reaction Scheme:

Experimental Details:

To the mixture of compound 1 (0.5 g, 1.0 eq), 3-trifluoromethyl phenylboronic acid (0.63 g, 1.0 eq), Na₂CO₃ (0.46 g, 1.5 eq) in 15 mL ofdioxane was added Pd(PPh₃)₄ (0.33 g, 0.1 eq), and the reaction mixturewas refluxed under N₂ for 16 h. After cooling, the mixture was filteredand the filtrate was evaporated to dryness and purified by columnchromatography to give compound 2.

2. Reaction Scheme:

Experimental Details:

A mixture of compound 2 (0.2 g, 1.0 eq), SeO₂ (0.19 g, 2.0 eq) in 10 mLof acetic acid was refluxed under N₂ for 48 h. Solvent was removed byevaporation and the residue was dissolved in water and adjusted to pH 6with saturated NaHCO₃ solution, extracted with dichloromethane. Theorganic layers were collected, dried and evaporated to dryness. Thecrude product was purified by column chromatography to give compound 3.

3. Reaction Scheme:

Experimental Details:

The mixture of compound 3 (30 mg, 1.0 eq) and compound 4 (19 mg, 1.0 eq)in 5 mL of dichloromethane was stirred at r.t. overnight. The mixturewas concentrated to dryness and purified by preparative HPLC to give thedesired compound.

Example 22

1. Reaction Scheme:

Experimental Details:

The mixture of compound 1 (0.5 g, 1.0 eq), trifluoromethyl phenylamine(0.58 g, 1.0 eq), EDC (1.05 g, 1.5 eq), HOBt (50 mg, 0.1 eq) in 15 mL ofdichloromethane was stirred at r.t. overnight. The mixture was washedwith 1 N NaOH solution, water, extracted with dichloromethane. Theorganic layer was dried over Na₂SO₄, concentrated to dryness, purifiedby column chromatography to give compound 2.

2. Reaction Scheme:

Experimental Details:

A mixture of compound 2 (0.2 g, 1.0 eq), SeO₂ (0.16 g, 2.0 eq) in 10 mLof acetic acid was refluxed under N₂ for 48 h. Solvent was removed byevaporation and the residue was dissolved in water and adjusted to pH 6with saturated NaHCO₃ solution, extracted with dichloromethane. Theorganic layers were collected, dried and evaporated to dryness. Thecrude product was purified by column chromatography to give compound 3.

3. Reaction Scheme:

Experimental Details:

The mixture of compound 3 (40 mg, 1.0 eq) and compound 4 (30 mg, 1.0 eq)in 5 mL of dichloromethane was stirred at r.t. overnight. Theprecipitates were collected and washed with dichloromethane, dried undervacuum to give the desired compound.

Example 23

1. Reaction Scheme:

Experimental Details:

The solution of compound 1 (3.0 g, 1.0 eq) and 2 mL 98% H₂SO₄ in 10 mLof EtOH was refluxed for 4 h, cooled to r.t., evaporated to dryness,diluted with water, adjusted to pH 8 with NaHCO₃, extracted withdichloromethane. The organic layer was dried and concentrated to givecompound 2.

2. Reaction Scheme:

Experimental Details:

A mixture of compound 2 (1.7 g, 1.0 eq), SeO₂ (2.29 g, 2.0 eq) in 80 mLof acetic acid was refluxed under N₂ for 48 h. Solvent was removed byevaporation and the residue was dissolved in water and adjusted to pH 6with saturated NaHCO₃ solution, extracted with dichloromethane. Theorganic layers were collected, dried and evaporated to dryness. Thecrude product was purified by column chromatography to give compound 3.

3. Reaction Scheme:

Experimental Details:

The solution of compound 3 (1.1 g, 1.0 eq), diethoxymethoxy-ethane (2.3g, 2.5 eq) and TsOH.H₂O (0.12 g, 0.1 eq) in 20 mL of ethanol wasrefluxed for 5 h. The solvent was evaporated and the solid was dissolvedin EtOAc, washed with water. The organic layer was dried over Na₂SO₄ andevaporated to give compound 4.

4. Reaction Scheme:

Experimental Details:

To the solution of compound 4 (0.6 g, 1.0 eq) in 10 mL of methanol wasadded 4 mL of 1 N NaOH solution, and the mixture was stirred at r.t.overnight. Solvent was evaporated and the residue was acidified to pH 6with 5% citric acid, extracted with dichloromethane. The organic layerwas dried, concentrated to give compound 5.

5. Reaction Scheme:

Experimental Details:

The mixture of compound 5 (0.4 g, 1.0 eq), trifluoromethyl phenylamine(0.29 g, 1.0 eq), EDC (0.51 g, 1.5 eq), HOBt (25 mg, 0.1 eq) in 10 mL ofdichloromethane was stirred at r.t. overnight. The mixture was washedwith 1 N NaOH solution, water, extracted with dichloromethane. Theorganic layer was dried over Na₂SO₄, concentrated to dryness, purifiedby column chromatography to give compound 6.

6. Reaction Scheme:

Experimental Details:

The solution of compound 6 (0.2 g, 1.0 eq) in 10 mL of dioxane wastreated with 4 mL of 1 N HCl, and the mixture was heated to 60° C. for 2h. After cooling, pH was adjusted to 8 by addition of NaHCO₃. Themixture was extracted with dichloromethane, washed the organic layerwith water, dried with Na₂SO₄ and evaporated to dryness. The crudeproduct was purified by column chromatography to give compound 7.

7. Reaction Scheme:

Experimental Details: The mixture of compound 7 (30 mg, 1.0 eq) andcompound 8 (19 mg, 1.0 eq) in 5 mL of dichloromethane was stirred atr.t. overnight. The precipitates were collected and washed withdichloromethane, dried under vacuum to give the desired compound.

Example 24

1. Reaction Scheme

Experimental Details:

A mixture of compound 1 (2.0 g, 1.0 eq), SeO₂ (2.6 g, 2.0 eq) in 80 mLof acetic acid was refluxed under N2 for 36 h. Solvent was removed byevaporation and the residue was dissolved in water and adjusted to pH 6with saturated NaHCO₃ solution, extracted with dichloromethane. Theorganic layers were collected, dried and evaporated to dryness. Thecrude product was purified by column chromatography to give compound 2.

2. Reaction Scheme:

Experimental Details:

The solution of compound 2 (0.5 g, 1.0 eq), diethoxymethoxy-ethane (1.0g, 2.5 eq) and TsOH.H₂O (0.05 g, 0.1 eq) in 8 mL of ethanol was refluxedfor 3 h. The solvent was evaporated and the solid was dissolved inEtOAc, washed with water. The organic layer was dried over Na₂SO₄ andevaporated to give compound 3.

3. Reaction Scheme:

Experimental Details:

To the mixture of compound 3 (0.6 g, 1.0 eq),3-trifluoromethyl-phenylamine (0.37 g, 1.0 eq), t-BuONa (0.26 g, 1.2 eq)in 15 mL of toluene was added under N₂ Pd₂(dba)₃ (42 mg, 0.02 eq) andxantphos (28 mg, 0.02 eq). The mixture was refluxed under N₂ for 16 h,cooled, filtered. The filtrate was concentrated and purified by columnchromatography to give compound 4.

4. Reaction Scheme:

Experimental Details:

The solution of compound 4 (0.25 g, 1.0 eq) in 10 mL of dioxane wastreated with 4 mL of 1 N HCl, and the mixture was heated to 60° C. for 2h. After cooling, the pH was adjusted to 8 by addition of NaHCO₃. Themixture was extracted with dichloromethane, washed the organic layerwith water, dried with Na₂SO₄ and evaporated to dryness. The crudeproduct was purified by column chromatography to give compound 5.

5. Reaction Scheme:

Experimental Details:

The mixture of compound 5 (30 mg, 1.0 eq) and compound 6 (19 mg, 1.0 eq)in 5 mL of dichloromethane was stirred at r.t. overnight. Theprecipitates were collected and washed with dichloromethane, dried undervacuum to give the desired compound.

Example 25

1. Reaction Scheme:

Experimental Details:

To a solution of compound 1 (14 g, 0.1 mol) in aqueous HBr (30 mL) wasadded a solution of NaNO (8.3 g 0.15 mol) in H₂O (10 mL) at 0° C. over aperiod of 30 min. After stirring for 60 min, the reaction mixture wasadded to a solution of CuBr (14 g, 0.1 mol) in aqueous HBr (16 mL) at80° C. After complete addition, the reaction mixture was stirred at thesame temperature for 2 h. After cooling to room temperature, thereaction mixture was extracted with EA (100 mL×3). The combined organiclayer were washed with brine and dried over Na₂SO₄. After filtrating offthe Na₂SO₄, the filtrate was concentrated to dryness. The residue waspurified by column to give the product 2.

2. Reaction Scheme:

Experimental Details:

To a stirred and degassed mixture of compound 2 (4.11 g, 0.02 mol) andcompound 3 (4.74 g, 0.02 mol), and KOH (5.28 g, 0.1 mol) and TBBA (6.44g, 0.02 mol) in anhydrous THF (100 mL) was added Pd (PPh₃)₄ (2.31 g, 2mmol) under N₂ atmosphere and stirred under reflux for 12 h. Afterfiltrating off the solid, the filtrate was concentrated to dryness. Theresidue was purified by column to give the product 4.

3. Reaction Scheme:

Experimental Details:

A solution of 4 (1 g, 3 mmol) in dichloromethane (10 mL) was treatedwith TFA (1 mL) and then stirred for 6 h at room temperature. Thesolvent was removed under reduced pressure to give the product 5 whichis done next step without purification.

4. Reaction Scheme:

Experimental Details:

To a stirred and degassed mixture of compound 5 (619 mg, 2.4 mmol) andcompound 6 (520 mg, 2.4 mmol), and _(t)BuONa (460 mg, 4.8 mmol) andBINAP (599 mg, 6.9 mol) in toluene (60 mL) was added Pd₂(dba)₃ (221 mg,0.024 mmol) under N₂ atmosphere and stirred at 80° C. for 48 h. Afterfiltrating off the solid, the filtrate was concentrated to dryness. Theresidue was purified by column to give the product 7.

5. Reaction Scheme:

Experimental Details:

A solution of compound 7 (396 mg, 0.1 mmol) in dichloromethane (10 mL)was treated with BBr₃ (146 mg, 0.6 mmol) at −30° C. under N₂ atmosphere,then was stirred at room temperature for 4 h. The reaction was pouredunto ice-water and then was brought by adding Na₂CO₃. The resultingmixture was extracted with dichloromethane (25 mL×3), the combinedorganic layer was dried over Na₂SO₄. After filtrating off the Na₂SO₄,the filtrate was concentrated to give the crude product 8, which wasdone next step without purification.

6. Reaction Scheme:

Experimental Details:

A solution of compound 8 (32.2 mg, 0.1 mmol) and compound 9 (21 mg, 0.1mmol) in anhydrous CH₂Cl₂ (300 mL) was stirred under reflux for 6 h. Thesolvent was removed under reduced pressure. The residue was separated byprep-TLC to give the desired compound.

Example 26

1. Reaction Scheme:

Experimental Details:

A solution of 5-fluoro-1H-pyrimidine-2,4-dione (113 g, 0.5 mol) inN,N-dimethylaniline (70 mL) was treated with POCl₃ (500 mL), then wasreflux for 2 h. After cooling to room temperature, the reaction mixturewas poured onto ice-water. The resulting mixture was extracted withethyl acetate (100 mL×3). The combined organic layers were washed withsaturated aqueous of NaHCO₃, then brine. The solvent was removed underreduced pressure to give compound 2.

2. Reaction Scheme:

Experimental Details:

To a solution of compound 2 (20.8 g, 0.194 mol) in ethanol (300 mL) wasadded morpholine (21.6 g, 0.25 mol) drop wise at −10° C. over a periodof 15 min. This mixture was stirred at room temperature for 0.5 h,heated then to 50° C. for 15 min. After cooling to room temperature anddilution with water, solid was precipitated. The solid was collected byfiltrate and washed with water to give compound 3.

3. Reaction Scheme:

Experimental Details:

A solution of 3 (4.6 g, 17.5 mmol) and hydrazine (8.75 g, 87.5 mmol) inethanol (40 mL) was heated to reflux for 6 h. After cooling andprecipitating, the precipitate was collected by filtrate and washed withethanol to give compound 4.

4. Reaction Scheme:

Experimental Details:

A solution of compound 5 (14 g, 50 mmol) in anhydrous THF (100 mL) wastreated with BuMgCl (37.5 mL, 60 mmol) at −15° C. under N₂ atmosphere.After complete addition, this mixture was stirred at this temperaturefor 1 h. Anhydrous DMF (0.54 g, 75 mmol) was added to the reactionmixture at 0° C. over a period of 30 min, warmed then to roomtemperature for 1 h. The reaction mixture was quenched by adding 2 M HCl(80 mL). The result mixture was extracted with ethyl acetate (50 mL×3).The combined organic layers were dried over Na₂SO₄. The solvent wasconcentrated to dryness. The residue was separated by column to givecompound 6.

5. Reaction Scheme:

Experimental Details:

A solution of compound 6 (4.5 g, 22.5 mmol) in triethyl orthoformate (15mL) was heated in the presence of a trace of TsOH over night. Thereaction mixture was diluted with ethyl acetate (100 mL) and washed withan aqueous of 5% Na₂CO₃. The organic layer was separated and dried overNa₂SO₄. The solvent was concentrated to give compound 7.

6. Reaction Scheme:

Experimental Details:

To a stirred and degassed mixture of compound 7 (1.3 g, 5 mmol) andcompound 8 (0.97 g, 6 mmol), and t BuONa (0.7 g, 7 mmol) and P(t-Bu)₃(15 mg) in toluene (60 mL) was added Pd₂(dba)₃ (23 mgl) under N2atmosphere and stirred under reflux for 12 h. After filtrating off thesolid, the filtrate was concentrated to dryness. The residue waspurified by column to give product 9.

7. Reaction Scheme:

Experimental Details:

A solution of compound 9 (200 mg, 0.58 mmol) in dichloromethane (10 mL)was treated with BBr₃ (146 mg, 0.6 mmol) at −30° C. under N₂ atmosphere,then was stirred at room temperature for 4 h. The reaction was pouredunto ice-water and then was brought by adding Na₂CO₃. The resultingmixture was extracted with dichloromethane (25 mL×3), the combinedorganic layer was dried over Na₂SO₄. After filtrating off the Na₂SO₄,the filtrate was concentrated to give the crude product 10.

8. Reaction Scheme:

Experimental Details:

A solution of compound 10 (48.7 mg, 0.2 mmol) and compound 4 (63 mg, 0.2mmol) in anhydrous CH₂Cl₂ (300 mL) was stirred under reflux for 6 h. Thesolvent was removed under reduced pressure. The residue was separated byprep-TLC to give the desired compound.

Example 27

1. Reaction Scheme:

Experimental Details:

A solution of 3 (2.4 g, 11 mmol) and methylhydrazine (2 g, 45 mmol) inethanol (40 mL) was heated to reflux for 6 h. After cooling andprecipitating, the precipitate was collected by filtrate and washed withethanol to give compound 2.

2. Reaction Scheme:

Experimental Details:

A solution of compound 2 (28 mg, 0.1 mmol) and compound 3 (37 mg, 0.1mmol) in anhydrous CH₂Cl₂ (300 mL) was stirred under reflux for 6 h. Thesolvent was removed under reduced pressure. The residue was separated byprep-TLC to give the desired compound.

Example 28

1. Reaction Scheme:

Experimental Details:

A solution of 1 (2 g, 0.011 mol) in anhydrous THF (100 mL) was treatedwith MeMgCl (15 mL. 0.038 mol), at −20° C. and stirred for 2 h at thistemperature. This reaction mixture was quenched by adding the saturatedaqueous of NH₄Cl. The resulting mixture was extracted with ethyl acetate(100 mL×3). The combined organic layers were washed with brine. Thesolvent was removed under reduced pressure to give compound 2.

2. Reaction Scheme:

Experimental Details:

A solution of compound 2 (2 g, 0.01 mol) and ethylene glycol (3 g, 0.048mol) in anline (100 mL) was heated in the presence of a trace of TsOHfor 3 h. The reaction mixture was diluted with ethyl acetate (100 mL)and washed with an aqueous of 5% Na₂CO₃. The organic layer was separatedand dried over Na₂SO₄. The solvent was concentrated to give compound 3.

3. Reaction Scheme:

Experimental Details:

To a stirred and degassed mixture of compound 3 (0.4 g, 1.6 mmol) andcompound 4 (0.3 g, 1.9 mmol), and _(t)BuONa (0.22 g, 2 mmol) andP(t-Bu)₃ (59 mg) in toluene (30 mL) was added Pd₂(dba)₃ (29 mgl) underN₂ atmosphere and stirred under reflux for 12 h. After filtrating offthe solid, the filtrate was concentrated to dryness. The residue waspurified by column to give product 5.

4. Reaction Scheme:

Experimental Details:

A solution of compound 5 (100 mg, 0.3 mmol) in dichloromethane (10 mL)was treated with BBr₃ (146 mg, 0.6 mmol) at −30° C. under N2 atmosphere,then was stirred at room temperature for 4 h. The reaction was pouredunto ice-water and then was brought by adding Na₂CO₃. The resultingmixture was extracted with dichloromethane (25 mL×3), the combinedorganic layer was dried over Na₂SO₄. After filtrating off the Na₂SO₄,the filtrate was concentrated to give the crude product 6.

5. Reaction Scheme:

Experimental Details:

A solution of compound 6 (64 mg, 0.2 mmol) and compound 7 (45 mg, 0.2mmol) in anhydrous CH₂Cl₂ (300 mL) was stirred under reflux for 6 h. Thesolvent was removed under reduced pressure. The residue was separated byprep-TLC to give the desired compound.

Example 29

1. Reaction Scheme:

Experimental Details:

A mixture of 1 (5.2 g, 22 mmol) and 2 (2.44 g, 20 mmol) in an aqueous of2 M Na₂CO₃ (25 mL) and toluene (40 mL) was stirred with Pd(PPh₃)₄ (0.57g, 0.05 mmol) under reflux over night. The reaction mixture wasextracted with ethyl acetate (100 mL×3). The combined organic layerswere washed with brine. The solvent was removed under reduced pressureto dryness. The residue was purified by column to give 3.

2. Reaction Scheme:

Experimental Details:

A solution of compound 3 (0.78 g, 3.3 mmol) and triisopropyl borate (1mL, 4 mmol) in anhydrous toluene (50 mL) was treated with n-BuLi (1.5mL, 3.75 mmol) at −60° C. under N2 atmosphere. After complete addition,the mixture was stirred at −10° C. for 1 h. The reaction mixture wasquenched by adding aqueous of 2 M HCl, and washed with toluene. Theaqueous layer was brought pH=8 by adding Na₂CO₃, extracted then withethyl acetate (50 mL×3). The combined organic layers were washed withbrine. The solvent was removed under reduced pressure to dryness. Theresidue was separated by column to give 4.

3. Reaction Scheme:

Experimental Details:

To a stirred and degassed mixture of compound 4 (2.8 g, 14 mmol) andcompound 5 (7 g, 42 mmol) in an aqueous of 2 M Na₂CO₃ (250 mL) andtoluene (40 mL) was stirred with Pd(PPh₃)₄ (0.57 g, 0.05 mmol) underreflux over night. The reaction mixture was extracted with ethyl acetate(100 mL×3). The combined organic layers were washed with brine. Thesolvent was removed under reduced pressure to dryness. The residue wasseparated by column to give 6.

4. Reaction Scheme:

Experimental Details:

A solution of 6 (0.53 g, 1.9 mmol) and hydrazine (0.52 g, 8.8 mmol) inethanol (50 mL) was stirred under reflux for 6 h. After cooling andprecipitating, the precipitate was collected by filtrate and washed withethanol to give compound 7.

5. Reaction Scheme:

Experimental Details:

A solution of compound 7 (53 mg, 0.13 mmol) and compound 8 (79 mg, 0.13mmol) in anhydrous CH₂Cl₂ (300 mL) was stirred under reflux for 6 h. Thesolvent was removed under reduced pressure. The residue was purified byprep-TLC to give the desired compound.

Example 30

1. Reaction Scheme:

Experimental Details:

A mixture of 1 (3.1 g, 13 mmol) and 2 (1 g, 12 mmpl) in an aqueous of 2M Na₂CO₃ (15 mL) and toluene (30 mL) was stirred with Pd(PPh₃)₄ (0.4 g,0.029 mmol) under reflux over night. The reaction mixture was extractedwith ethyl acetate (100 mL×3). The combined organic layers were washedwith brine. The solvent was removed under reduced pressure to dryness.The residue was separated by column to give 3.

2. Reaction Scheme:

Experimental Details:

A solution of compound 3 (2.0 g, 10 mmol) and triisopropyl borate (7 mL,30 mmol) in anhydrous toluene (50 mL) was treated with n-BuLi (12 mL, 30mmol) at −60° C. under N₂ atmosphere. After complete addition, thismixture was stirred at −10° C. for 1 h. The reaction mixture wasquenched by adding an aqueous of 2 m HCl, and washed with toluene. Theaqueous layer was brought pH=8 by adding Na₂CO₃, extracted then withethyl acetate (50 mL×3). The combined organic layers were washed withbrine. The solvent was removed under reduced pressure to dryness. Theresidue was separated by column to give 4.

3. Reaction Scheme:

Experimental Details:

To a stirred and degassed mixture of compound 4 (0.5 g, 3 mmol) andcompound 5 (1.5 g, 9 mmol) in an aqueous of 2 M Na₂CO₃ (3.5 mL) andtoluene (40 mL) was stirred with Pd(PPh₃)₄ (94 mg, 0.003 mmol) underreflux over night. The reaction mixture was extracted with ethyl acetate(100 mL×3). The combined organic layers were washed with brine. Thesolvent was removed under reduced pressure to dryness. The residue waspurified by column to give 6.

4. Reaction Scheme:

Experimental Details:

A solution of 6 (0.24 g, 1 mmol) and hydrazine (0.3 g, 4.7 mmol) inethanol (50 mL) was stirred under reflux for 6 h. After cooling andprecipitating, the precipitate was collected by filtrate and washed withethanol to give compound 7.

5. Reaction Scheme:

Experimental Details:

A solution of compound 7 (70 mg, 0.29 mmol) and compound 8 (83 mg, 0.3mmol) in anhydrous CH₂Cl₂ (300 mL) was stirred under reflux for 6 h. Thesolvent was removed under reduced pressure. The residue was purified byprep-TLC to give the desired compound.

Example 31

1. Reaction Scheme:

Experimental Details:

To a solution of compound 2 (0.83 g, 5 mmol) in ethanol (100 mL) wasadded benzylamine (0.54 g, 5 mmol) drop wise. After stirring for 2 h,the reaction mixture was diluted with water. The resulting mixture wasextracted with ethyl acetate (50 mL×3). The combined organic layers weredried over Na₂SO₄. The solvent was concentrated to give compound 3.

2. Reaction Scheme:

Experimental Details:

A solution of 3 (1.18 g, 5 mmol) and hydrazine (5 ml) in ethanol (40 mL)was heated to reflux for 6 h. After cooling and precipitating, theprecipitate was collected by filtrate and washed with ethanol to givecompound 4.

3. Reaction Scheme:

Experimental Details:

A solution of compound 4 (48.7 mg, 2 mmol) and compound 5 (63 mg, 0.2mmol) in anhydrous CH₂Cl₂ (300 mL) was stirred under reflux for 6 h. Thesolvent was removed under reduced pressure. The residue was separated byprep-TLC to give the desired compound.

Example 32

1. Reaction Scheme:

Experimental Details:

To a solution of compound 2 (0.83 g, 5 mmol) in ethanol (100 mL) wasadded compound 2 (0.35 g, 5 mmol) dropwise. After stirring for 2 h, thereaction mixture was diluted with water. The resulting mixture wasextracted with ethyl acetate (50 mL×3). The combined organic layers weredried over Na₂SO₄. The solvent was concentrated to give compound 3.

2. Reaction Scheme:

Experimental Details:

A solution of 3 (1.0 g, 5 mmol) and hydrazine (5 mL) in ethanol (40 mL)was heated to reflux for 6 h. After cooling and precipitating, theprecipitate was collected by filtrate and washed with ethanol to givecompound 4.

3. Reaction Scheme:

Experimental Details:

A solution of compound 4 (480 mg, 2 mmol) and compound 5 (60 mg, 0.2mmol) in anhydrous CH₂Cl₂ (300 mL) was stirred under reflux for 6 h. Thesolvent was removed under reduced pressure. The residue was separated byprep-TLC to give the desired compound.

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What is claimed is:
 1. A method for determining whether a substance is aspecific inhibitor of a protein that is capable of eliciting adetectable phenoresponse, which comprises: a) incubating a test cellwhich expresses the protein and is capable of eliciting a phenoresponselinked to the presence and functional activity of the protein in thecell with the substance; b) incubating a control cell which expressesthe protein at a lower level or does not express the protein and iscapable of eliciting a detectable phenoresponse linked to the presenceand functional activity of the protein in the cell to a lesser extent ornot at all; c) comparing the phenoresponse of the test cell treated withthe substance to the phenoresponse of the control cell treated with thesubstance; and d) determining that the substance is a specific inhibitorof the protein if the substance is capable of modulating thephenoresponse of the test cell to a greater extent than the controlcell.
 2. A method for determining whether a substance is a specificactivator of a protein that is capable of eliciting a detectablephenoresponse, which comprises: a) incubating a test cell whichexpresses the protein and is capable of eliciting a phenoresponse linkedto the presence and functional activity of the protein in the cell withthe substance; b) incubating a control cell which expresses the proteinat a lower level or does not express the protein and is capable ofeliciting a detectable phenoresponse linked to the presence andfunctional activity of the protein in the cell to a lesser extent or notat all; c) comparing the phenoresponse of the test cell treated with thesubstance to the phenoresponse of the control cell treated with thesubstance; and d) determining that the substance is a specific activatorof the protein if the substance is capable of modulating thephenoresponse of the test cell to a greater extent than the controlcell.